Methods and apparatus for centrifugal liquid chromatography

ABSTRACT

Apparatus and methods related to centrifugal liquid chromatography are described. An angular velocity can be simultaneously imparted to a large number of chromatographic enclosures. Via centrifugal forces, a mobile phase fluid including a sample can be driven through a stationary phase within the chromatographic enclosure to perform a chromatographic separation process on components of the sample. The use of centrifugation as a driving force can allow significantly smaller stationary phase particles to be employed as compared to high performance liquid chromatography (HPLC). Further, for an equivalent chromatographic separation process, the use of centrifugation can provide much greater separation efficiencies than HPLC.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application Ser. No. 61/210,118, entitled“Centrifugal Column Chromatograph System,” by Kerr et al. filed Mar. 13,2009, which is incorporated herein in its entirety and for all purposes.This application also claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application Ser. No. 61/313,215, entitled “Methodsand Apparatus for Centrifugal Liquid Chromatography,” by Kerr et al.filed Mar. 12, 2010, which is incorporated herein in its entirety andfor all purposes.

FIELD OF THE INVENTION

The described embodiments relate generally to systems for performingliquid chromatography. More particularly, the present embodiments relateto methods and apparatus for performing liquid chromatography in acolumn subject to centrifugal forces.

DESCRIPTION OF THE RELATED ART

Chromatography is a tool for practicing chemists as well as others whoare applying chemistry in their own discipline. Chromatography is widelyused in and essential to the petroleum industry, food industry,pharmaceutical industry, medicine (e.g., diagnostics), and many others.Chromatography is a separation process. During the chromatographicprocess, the constituents of a mixture can be physically separated fromone another. The physically separated constituents can be analyzed, suchas for the purposes of identification. Also, the separated constituentscan be collected and utilized for other purposes, such as for use as acomponent in forming another chemical composition.

Two important aspects of chromatography are separation efficiency andthroughput. In the chromatographic process, separation efficiencyrelates to the ability of the process to separate constituents of amixture such that the constituents can be distinguished from one anotherand their chemical identity can be established and collected if desired.A consequence of inadequate separation efficiency in a chromatographicprocess is an inability to either identify or collect particularconstituents of the mixture on which the chromatographic process isbeing performed. Throughput relates to how long a chromatographicprocess takes and when a separated constituent is collected, how long ittakes to gather a particular amount of the constituent. Typically, asthroughput times increase the costs of utilizing a chromatographicprocess and an associated chromatographic system increase.

A commonly used methodology to perform chromatographic separation ishigh-performance liquid chromatography (HPLC). Currently, in liquidchromatography, chromatographic systems employing HPLC provide some ofthe best separation efficiencies and throughput times. HPLC usespressure as a driving force to move a liquid including a mixture ofconstituents to be separated through a bed of particles. When a liquidis moved through the bed of particles, interactions between the bed ofparticles and the liquid can separate the constituents of the liquidfrom one another.

To improve separation efficiency, it is desirable to use smallerparticle sizes within the bed of particles. However, as the particlesize decreases, throughput times can increase if the liquid is notefficiently moved through the bed of particles. In HPLC, further gainsin separation efficiency are currently limited by the use of pressure asa driving force. To employ a particle size of about 2 micrometers indiameter and obtain a sufficient throughput time, in HPLC, pressures ofabout 10,000 PSI are required. It is expected that increasing pressuresbeyond this level will be of limited benefit because at greater pressurelevels the particles tend to be crushed and the costs associated withconstructing and maintaining a system that provides the greater pressurelevels are prohibitive. Therefore, there is a need for improved methodsand apparatus of performing liquid chromatography that can providebetter separation efficiency and throughput without the limitations ofHPLC.

SUMMARY OF THE DESCRIBED EMBODIMENTS

This paper describes various embodiments that relate to systems,methods, and apparatus for providing centrifugal liquid chromatography.A rotor with one or more chromatographic enclosures is provided. Eachchromatographic enclosure can be arranged to contain a chromatographicstationary phase and to provide a flow path through the chromatographicstationary phase. Via centrifugal forces, a mobile phase fluid includinga sample can be driven through the chromatographic stationary phasewithin the chromatographic enclosure to perform a chromatographicseparation process on components of the sample. Introduction of thesample can be controlled to allow a flow on the rotor to reach asteady-state condition prior to sample introduction. The use ofcentrifugation as a driving force can allow significantly smallerstationary phase particles to be employed as compared to highperformance liquid chromatography (HPLC). Further, for an equivalentchromatographic separation process, the use of centrifugation canprovide much greater separation efficiencies than HPLC.

One aspect is generally characterized as a centrifugal chromatographicsystem. The centrifugal chromatographic system includes 1) achromatographic enclosure; 2) a rotor that carries the chromatographicenclosure where the rotor is configured to rotate the chromatographicenclosure; and 3) a sample introduction mechanism in fluid communicationwith the chromatographic enclosure. The sample introduction mechanism isarranged to introduce a sample fluid to the chromatographic enclosurewhile the rotor is rotating. Further, the sample introduction mechanismis configured to receive a sample introduction signal and to trigger theintroduction of the sample fluid in response to receiving the sampleintroduction signal. In a particular embodiment, a controller is coupledto the sample introduction mechanism where the controller is arranged toautomatically trigger the introduction of the sample fluid. In variousembodiments, the sample introduction mechanism and/or the controller canbe carried on the rotor.

Another aspect is generally characterized as centrifugal columnchromatographic system. The centrifugal column chromatographic systemincludes 1) a rotor configured to rotate about an axis; 2) a number ofchromatographic column enclosures carried by the rotor and 3) a sampleintroduction mechanism in fluid communication with at least a selectedone of the chromatographic column enclosures and 4) an eluent reservoircarried by the rotor, in fluid communication with the number ofchromatographic enclosures for receiving fluids eluted from theplurality of chromatographic enclosures.

In various embodiments, each chromatographic column enclosure isarranged to contain an associated chromatographic stationary phase andto facilitate transmission of a fluid through the chromatographicstationary phase contained within the chromatographic column enclosure.The fluid is driven generally axially through the chromatographic columnenclosure including through the chromatographic stationary phase viacentrifugal force generated from the rotation of the rotor. The sampleintroduction mechanism is arranged to introduce a sample fluid to theselected chromatographic column enclosures while the rotor is rotating.

Yet another aspect is generally characterized as a centrifugalchromatographic system. The centrifugal chromatographic systemincludes 1) a rotor configured to rotate about an axis; 2) at least onechromatographic enclosure carried by the rotor; and 3) a mixing chambercarried by the rotor and arranged to mix a mobile phase fluid with asample on the rotor to create a mixed fluid. The mixing chamber utilizesthe rotation of a component carried by the rotor to enhance the mixingof the sample with the mobile phase fluid. Further, the mixing chamberis typically located upstream of the chromatographic enclosures. Invarious embodiments, the centrifugal chromatographic system can alsoinclude a mobile phase fluid reservoir in fluid communication with themixing chamber; and a sample introduction mechanisms in fluidcommunication with the mixing chamber where the mobile phase fluidreservoir and the sample introduction mechanism may be located either onor off the rotor.

A further aspect is generally characterized as a chromatographic system.The chromatographic system can include 1) a chromatographic enclosurewhere chromatographic enclosure is arranged to contain an associatedchromatographic stationary phase and to facilitate transmission of afluid through the chromatographic stationary phase contained within thechromatographic enclosure; 2) a rotor that carries the chromatographicenclosure wherein the rotor is configured to rotate the chromatographicenclosure at an angular velocity such that fluid is driven throughchromatographic enclosure including through the chromatographicstationary phase via centrifugal force; and 3) a fluid enclosureincluding a first portion configured to remain stationary while therotor is rotating and a second portion, carried on the rotor, configuredto rotate with the rotor wherein the fluid enclosure is in fluidcommunication with the chromatographic enclosure.

In one embodiment, the chromatographic system can include a fluiddelivery mechanism in fluid communication with the chromatographicenclosure configured to facilitate the delivery of a mobile phase fluidto the chromatographic enclosure. The fluid delivery mechanism includesa first portion configured to remain stationary while the rotor isrotating and a second portion carried on the rotor and configured torotate with the rotor. The fluid delivery mechanism is arranged so thatfluid can be passed from the first portion to the second portion of thefluid delivery mechanism while the rotor is rotating.

One aspect is generally characterized as a centrifugal chromatographicsystem. The centrifugal chromatographic system includes 1) a rotorincluding a chromatographic enclosure carried by the rotor; and 2) a gasbearing proximate to the rotor wherein the gas bearing is arranged tostabilize the rotor while it is rotating. The system can also include acontainment structure surrounding the rotor; and a plurality of rotorsupport structures that are supported by the containment structure. Therotor support structures can each include a gas bearing where the gasbearings cooperate to help stabilize the rotor while the rotor isrotating

An additional aspect is generally characterized as chromatographicsystem. The system can include 1) a plurality of chromatographicenclosures, carried on the rotor, each chromatographic enclosure beingarranged to contain an associated chromatographic stationary phase andto facilitate transmission of a fluid through the chromatographicstationary phase contained within the chromatographic enclosure wherethe fluid is driven through the chromatographic stationary phase andtowards an outer perimeter of the rotor via centrifugal force generatedfrom the rotation of the rotor; and 2) a plurality of links arrangedaround the outer perimeter of the rotor each link configured forconnection with two other links to form an unbroken chain to be aroundthe outer perimeter of the rotor; wherein one or more of the linksincludes a flow path arranged to i) receive the fluid moving toward theouter perimeter and ii) redirect the fluid inwardly away from the outerperimeter.

Another aspect is generally characterized as a centrifugalchromatographic system. The system includes 1) a rotor configured torotate about an axis; 2) a chromatographic enclosure carried on therotor, said chromatographic enclosure being arranged to contain anassociated chromatographic stationary phase and to facilitatetransmission of a fluid through the chromatographic stationary phasecontained within the chromatographic enclosure, wherein the fluid isdriven through the chromatographic stationary phase via centrifugalforce generated from the rotation of the rotor; and 3) a flow cell influid communication with the chromatographic enclosure, the flow cellincluding a flow window, wherein an inner cross sectional area of a flowpath through the chromatographic enclosure and the flow cell issubstantially constant starting at a location in the chromatographicstationary phase of the chromatographic enclosure and progressing pastthe flow window.

A further aspect can be generally characterized as method of operating acentrifugal chromatographic system. The method includes 1) rotating arotor that carries a chromatographic column; 2) establishing a flow of amobile phase fluid through the chromatographic column in whichcentrifugal force drives the mobile phase through the chromatographiccolumn; and 3) introducing a sample fluid into the chromatographiccolumn by inserting the sample fluid into the mobile phase fluid flowwhere the introduction of the sample occurs while the rotor rotates. Themethod can also include determining whether a steady mobile phase fluidflow condition has been reached within the chromatographic column wherethe sample fluid is released after it is determined that a steady mobilephase fluid flow condition has been reached within the chromatographiccolumn. After it is determined that a steady mobile phase fluid flowcondition has been reached, the method can further include introducing asample fluid into the chromatographic column to facilitate centrifugalchromatographic separation of the sample fluid.

An additional aspect can be generally characterized as a method ofoperating a centrifugal chromatographic system. The method includes 1)rotating a rotor that carries a chromatographic column that contains achromatographic stationary phase; 2) delivering a mobile phase fluid tothe chromatographic column while the rotor rotates so that centrifugalforce drives the mobile phase fluid through the chromatographicstationary phase; and 3) determining when to introduce a sample fluidinto the chromatographic column to facilitate centrifugalchromatographic separation of the sample fluid.

Other aspects and advantages of the invention will become apparent fromthe following detailed description taken in conjunction with theaccompanying drawings which illustrate, by way of example, theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be readily understood by the following detaileddescription in conjunction with the accompanying drawings, wherein likereference numerals designate like structural elements, and in which:

FIG. 1 is a block diagram of a chromatographic system including arotatable element.

FIG. 2 is a side view of an embodiment of a rotor assembly configuredfor use in a chromatographic system.

FIG. 3 is a top view of an embodiment of a rotor assembly configured foruse in a chromatographic system.

FIGS. 4A and 4B are a top perspective view and a bottom view of amanifold assembly configured for use in a chromatographic system.

FIG. 5A is a side cross sectional view of a rotor assembly with anillustrated flow path.

FIG. 5B is a top perspective cross sectional view of a platter,chromatographic column, a flow cell, a return segment link and a returnflow channel with an illustrated flow path.

FIG. 5C is a perspective view of a return segment link.

FIG. 6 is a side view of the rotor assembly with a cross-sectional viewof a reservoir.

FIGS. 7A-7C are side and perspective views of a gas bearing supportassembly.

FIG. 8 is side view of a rotor assembly, gas bearing assembly within acontainment structure and including instrument mounts.

FIG. 9A is a top view of an embodiment of a rotor assembly configuredfor use in a chromatographic system.

FIG. 9B is a side view of an embodiment of a rotor assembly configuredfor use in a chromatographic system.

FIG. 9C is a block diagram including a reaction chamber located betweenthe end of a column and a flow cell.

FIG. 10A is a front and side view of a bucket assembly including aplurality of columns for performing chromatographic separation.

FIG. 10B is a top view of a rotor assembly including a plurality ofbuckets during rotation.

FIG. 10C is a top view of a rotor assembly including a plurality ofbuckets at rest.

FIG. 10D is a top view of a rotor assembly including a plurality ofbuckets during rotation.

FIG. 11 is a front view of a column configured for a chromatographicprocess before and after centrifugation.

FIG. 12 is a chromatogram for a separation of 3 species.

FIG. 13 is a flow chart of a method for performing a chromatographicseparation process.

DETAILED DESCRIPTION OF SELECTED EMBODIMENTS

Reference will now be made in detail to representative embodimentsillustrated in the accompanying drawings. It should be understood thatthe following descriptions are not intended to limit the embodiments toone preferred embodiment. To the contrary, it is intended to coveralternatives, modifications, and equivalents as may be included withinthe spirit and scope of the described embodiments as defined by theappended claims.

Chromatography can be described as a process that achieves physicalseparation of the individual components of a mixture of chemicalsubstances. In the chromatographic process, the mixture of chemicalsubstances can be dissolved in (or mixed with) a carrier stream (gas orliquid). The carrier stream including the mixture can be forced througha bed of particles. The carrier stream moves at a velocity through thebed of particles. In chromatography, the carrier stream is oftenreferred to as the “mobile phase” and the bed of particles is referredto as the “stationary phase.”

The particles of the stationary phase can be selected such that thecomponents of the mixture dissolved in the mobile phase interactdifferently with the particles of the stationary phase. Differences inthe way components of the mixture interact with the particles can affecthow fast each component of the mixture moves through the stationaryphase. Thus, the components of the mixture can appear to be movingthrough stationary phase at different velocities. When two components ofthe mixture are moving through the stationary phase at differentvelocities, after traveling through the stationary phase for somedistance, the components can be become separated as the component withthe faster velocity through the stationary phase travels farther in agiven amount of time than the component with the lower velocity travelsthrough the stationary phase.

In liquid chromatography, an objective is to provide a system includinga selected mobile phase and a selected stationary phase that allows thecomponents of a mixture to move through the stationary phase atdifferent velocities. When the components of the mixture are separated,at least to some degree, various types of measurement devices can beused to characterize the properties of the separated components and insome instances determine a composition of the mixture. Further, ifdesired, components separated from the mixture can be collected forfurther processing and/or analysis.

In liquid chromatography, as described above, the mobile phase can beforced through the stationary phase, which can comprise particles, suchas spherical particles of a given radius. The particles can have poresand other molecules can be bound to the particles. The particles radius,pore size and molecules that bound to the particles can be selected tohave different types of physical interactions with components dissolvedin the mobile phase.

The earliest implementations of liquid chromatography used gravity as adriving force to move the mobile phase through the stationary phase. Forinstance, a mobile phase can be added to the top of an open columnpacked with particles of relatively uniform particle size distributionthat form a stationary phase. As the mobile phase moves through thestationary phase under the force of gravity, the components of themixture dissolved in the mobile phase can be separated. In someinstances, the separation of components can be observed visually asseparated components can have different colors. When the column is atransparent material, such as glass, for certain mixtures, bands ofdifferent colors can be observed moving down the column. A column packedwith particles and used to separate components in a mixture in thismanner can be referred to as a “chromatographic column.”

As liquid chromatography developed as a field, it was determined thatthe ability to separate components in a mixture using a chromatographiccolumn can be increased by decreasing the size of the packed particlesof the stationary phase. Using smaller particles increases the packingefficiency of the stationary phase and increases the surface area in agiven volume on which interactions can occur between the components ofthe mixture and the particles of the stationary phase. Thus, methods formanufacturing spherical particles with smaller dimensions for use in astationary phase of a chromatographic column were developed.

In a gravity-driven chromatographic column, the size of the particles inthe stationary phase can not be reduced indefinitely There exists aparticle size limit for which the force of gravity becomes insufficientto drive mobile phase and the mixture of components being analyzedthrough the stationary phase in a practical time frame. To overcome thelimits of using gravity as a driving force, other forces, such aspressure, can be used to move the mobile phase through thechromatographic column.

High performance liquid chromatography (HPLC) was developed to enablethe use of particles of smaller dimensions to be used in the stationaryphase which increases the separation efficiency of the chromatographiccolumn. In HPLC, high pressures are used as a driving force to move themobile phase through the chromatographic column. In HPLC, the pressureneeded to move the mobile phase through the stationary phase of achromatographic column is inversely proportional to the particlediameter squared. To use a particle size of a few microns, the HPLCsystem has to be configured to operate at pressures of about 10,000 PSI,which entails the use of high performance and, hence, expensivecomponents. Currently, pressure requirements appear to be a limitingfactor to further advances in HPLC, i.e., the use of smaller particlesin the stationary phase to increase separation efficiency.

To overcome the deficiencies related to pressure requirements of an HPLCsystem, it can be asked whether other forces can be used to drive amobile phase through a stationary phase in a chromatographic column. Asdescribed above, gravity was used in early chromatographic systems as adriving force. The effects of gravity on an object can be simulated andeven enhanced by placing the object on a platform, such as a disc, thatis revolving around an axis of rotation. From the perspective of theobject being moved in the rotational reference frame, the objectexperiences a force along a line that is perpendicular to the axis ofrotation that acts in a manner similar to gravity.

The force experienced by an object in a rotating reference frame can bereferred to as “centrifugal force.” Centrifugal force is oftencharacterized as some multiplier times the force of gravity. Forinstance, relative centrifugal force (RCF) is the measurement of theacceleration applied to an object being rotated around an axis and ischaracterized in units of “gravity” or g's. RCF can be calculated as,

RCF=r(2πN)² /g

where r is rotational radius, i.e., the distance of the object from therotational axis, N is the rotational speed, measured in revolutions perunit of time and g is earth's gravitational acceleration. RCF can alsobe written as,

RCF=1.118×10⁻⁵ r _(cm) N ² _(RPM)

where r is rotational radius measured in (cm) and N² _(RPM) is therotational speed measured in revolutions per minute (RPM).

As is described herein, RCF can be used as a driving force for moving amobile phase through a stationary phase packed in a chromatographiccolumn. To use RCF, a mobile phase can be passed through a stationaryphase in a chromatographic column while the chromatographic column isrotating. For example, chromatographic column can be placed on arotatable disc and chromatographic processes can be performed while thecolumn rotates. Devices and methods for using RCF in a chromatographicsystem are described with respect to the following figures.

First, a chromatographic system with one or more rotatable elements isdescribed with respect to FIG. 1. Then, details of a various embodimentsof a chromatographic system are described with respect to FIGS. 2-13. Inparticular, embodiments of a rotor assembly for a chromatographic systemare described with respect to FIGS. 2-9C. The embodiments of the rotorassembly can support and provide flow paths for a large number ofchromatographic enclosures during rotation. A “swinging bucket”configuration is described with respect to FIGS. 10A-10D. In the“swinging bucket” configuration the chromatographic enclosure is moreeasily detachable from the rotor assembly as compared to the embodimentsdescribed with respect to FIGS. 2-9C. An embodiment of a chromatographicenclosure before and after centrifugation is described with respect toFIG. 11. A chromatogram is described with respect of FIG. 12. Finally, amethod for performing a chromatographic separation process is describedwith respect to FIG. 13.

Chromatographic System

FIG. 1 is a block diagram of a chromatographic system 100 including arotatable element 132. In various embodiments, the rotatable element canalso be referred to as a “rotor.” The rotatable element 132 can be usedto apply RCF to components of the chromatographic system. Prior todiscussing the use and associated effects of RCF in a chromatographicsystem, such as 100, some elements of the chromatographic system 100 aredescribed. The description and number of elements in the chromatographicsystem are not meant to be limiting for the embodiments described hereinand are provided for the purposes of illustration only. Embodiments ofchromatographic systems with rotatable elements can have othercomponents as well as a different arrangement of components than thoseshown in FIG. 1.

The chromatographic system 100 can include solvent management 108. Thesolvent management 108 allows for different solvents to be used duringvarious chromatographic processes and can vary from run to run of thechromatographic system. The solvent management 108 can include solventreservoirs, such as 102. The solvent reservoirs can be used to provide abasis for a mobile phase in which a sample can be dissolved forchromatographic processing. A single solvent or a combination ofsolvents can be used depending on what type of sample is being analyzed.Further, the solvents that are used can be varied from run to run. Thus,a first solvent can be used for chromatographic analysis of a firstsample in a first run and a second solvent can be used forchromatographic analysis of a second sample in a second run. Flowcontrol mechanisms, such as valves, can be used to allow differentsolvents to be accessed at different times according to the requirementsof a particular run.

A solvent delivery system, such as 104, can be used to move solvent fromthe solvent reservoirs for the purposes of forming a mobile phase.Typically, a pump or pumps of some type can be used to provide a drivingforce for moving solvents from the reservoirs 102. The gradient former106 can be used to generate a mobile phase comprising a single solventor a combination of solvents. Binary solvent mixtures are commonly usedbut more complex solvent mixtures can be employed in the embodimentsdescribed herein. During a chromatographic run, when a mobile phase isformed from a combination of solvents, the concentrations of eachsolvent can be varied as a function of time. The gradient former 106 canbe used to control the variation in the concentrations of theconstituents of the mobile phase as a function of time.

The chromatographic system 100 can include sample management 114. Thesample management 114 allows for different to samples to be introducedto a mobile phase prior to introduction of the mobile phase into achromatographic column. The sample management 114 can provide storagefor various samples, such as 110, and mechanisms for loading the samplesinto a sample injector 112. The sample injector is an example of asample introduction mechanism. The sample injector, such as 112, can beused to introduce a selected sample into a mobile phase. For instance,the sample injector can inject the sample into the flow of a mobilephase moving in conduit.

The chromatographic system 100 can include column management 117. Thecolumn management can be used to control conditions, such as atemperature, associated with chromatographic columns, such as 116. Thetemperature of a chromatographic column can be controlled using a devicewhich either heats or cools the column such as a thermoelectric device(Peltier effect device) or any other method of adding or removingthermal energy from the column. Temperature gradients across a columncan result in different viscosities across the column and hence avelocity profile that varies across the column. Typically, thetemperature is highest near the center of the column and drops offtowards the walls of the column as a result of heat conduction throughthe walls. The walls of the column can be heated to reduce the change intemperature from the center of the column to the walls such that thetemperature uniform throughout the column. Also, temperature iswell-known to dramatically affect chemical equilibria such as theequilibrium between the analyte, the mobile phase and the stationaryphase. To obtain the most reproducible chromatographic results it can beimportant to have a stable temperature within the column.

The column management 117 can also include software for keeping track ofproperties of each column, such as but not limited to 1) when it waspacked, 2) a composition of the packing, i.e., stationary phase, 3) howmany times the column has been used and 4) characteristics of the typesof chromatographic runs in which it has been used, such as a compositionof the mobile phase solvents used from run to run.

The chromatographic system 100 can include detector management 120. Thedetector management 120 can involve controlling various instruments usedto characterize components separated from the mobile phase duringchromatographic processing in the chromatographic column. For example,one or more spectrophotometric detectors can be used to detect changesin the light intensity emitted from a light source (in the ultravioletand visible ranges, 190 nm to 700 nm), as the light passes throughwindows of a flow cell through which the column effluent is passing. Thephysically separated components of the mixture, still dissolved in themobile phase can pass through the flow cell where light emitted from afirst light source is passed through a first window of the flow cellsuch that it can interact with the components. Light can exit the flowcell through a second window of the flow cell near the second windowwhich can be gathered. The gathered light can be used to determinewhether an interaction between the light and one of the separatedcomponents has occurred.

The detector management 120 can output information to data management115. The data management 115 can be configured to collect, analyze andstore data derived from one or more detectors. The data management 115can also be configured to output data derived from a detector. Forinstance, the data management 115 can be configured to output achromatogram to a visual display associated with the chromatographicsystem. The data management 115 can be configured to track and storeinformation gathered from different chromatographic columns where thedata can be associated with various parameters of a particularchromatographic run.

The chromatographic system 100 can include collection management 124.The collection management can include fraction collectors 122 thatcollect separated components that have passed through the columns 116.Different components of interest can exit the column at different timesand the collection management 124 can be configured to route each of twoor more components to separate fraction collectors. Some components(components can be referred to as elutes) that exit the mobile phase maynot be of interest and can be considered as “waste.” For instance, priorto introducing of sample, a solvent can be passed through the column.The collection management 124 can collect the solvent that elutes fromthe column prior to the introduction of the sample as waste incollection device, such as waste collectors 123.

In the embodiments described herein, the chromatographic system cancomprise one or more rotatable elements, such as rotatable element 132.The rotatable elements can be controlled by the rotor management 140.The rotor management 140 can implement a rotation rate versus timeprofile for the rotatable element 132 including a spin up, a steady spinrate and a spin down. The rotor management 140 can monitor the rotatableelement 132 to ensure it is operating properly and perform proceduresassociated with operating the rotatable element 132, such asauto-balancing. The rotor management 140 can also monitor and controlpower delivery for various components operating on the rotatable element132, such as electronically controlled valves located on the rotatableelement.

The system management 103 can be configured to monitor and control theover-all functioning of the chromatographic system 100 during differentoperational modes of the system 100, such as initialization mode, anoperational mode and a shutdown mode. The system management 103 can beconfigured to communicate with and send commands to the flow management101, the solvent management 108, the sample management 114, the datamanagement 115, the column management 117, the detector management 120,the collection management 124 and the rotor management 140. The systemmanagement 103 can also be configured to communicate with other devicesand systems, such as other chromatographic systems and remote computers.

The chromatographic system 100 can involve a management of one or moreflows. The flow management 101 can be configured to coordinate flowthroughout the system 100. The flow management 101 can be configured tocontrol various valves and pumps located throughout the system 100directly or through communications with other device components. Forinstance, the flow management 101 can be configured to send a command tothe solvent management 108 to deliver a particular solvent flow rate andone or more logic devices associated with the solvent management 108 cancontrol devices, such as valves and pumps to the deliver commanded flowrate or the flow management 101 can be configured to directly controlthe valves and pumps associated with the solvent management 108.

Flow Path Management and Flow Analysis

The flow management system 101 can involve the establishment andmaintaining of multiple flow paths where the number of flow paths canvary from system to system or can be varied within one system. A flow inthe chromatographic system 100 can involve a start of the flow path 126,which as an example can start in the solvent reservoirs 102. Fluid canbe moved from the reservoirs to the gradient former 106 via the solventdelivery system 104. At a point 134 in the flow path, the flow can betransferred to the rotatable element 132. The transfer of flow can occurwhile the rotatable element 132 is rotating with a rotational velocityand a direction of rotation 138. In different embodiments, therotational velocity and direction of rotation can vary. In otherembodiments, the transfer of flow can occur while the rotatable elementis stationary.

On the rotatable element, 132, the flow at different locations can bemoving away from the center of the rotatable element 132 and at otherlocations, the flow can be moving toward the center of the rotatableelement 132. Along a flow path on the rotor, the flow can also be movedbetween different levels of the rotatable elements. For instance, theflow can move through chromatographic columns at a first level and thencan be moved to another level, such as to a reservoir located at a levelbelow the chromatographic columns.

In a particular embodiment, the flow can enter near a center of therotatable element and then start to flow away from the center and enterchromatographic columns, such as 116 a, 116 b and 116 c. For example,the flow can start with a common source near the center, such as amixing chamber located near the center, and then can be split intomultiple flow paths. For instance, the flow can be split during thesample management 114 to allow for different samples to be introducedinto different flow paths. This example of a split location is providedfor the purposes of illustration only. In particular embodiments, asplit location where a flow is branched into multiple flows can beimplemented at any point in the flow path, such within the solventmanagement 108, i.e., at the reservoirs 102, before or within solventdelivery 104, before or within the gradient former 106, before withinsample management 114, before or within the columns 116, before orwithin the detector management 120 or before or within the collectionmanagement 124. Further, the flow management 101 can be configured toimplement split locations that can vary from run to run of thechromatographic system.

As an example of flow splitting, the flow can start with a single flowpath prior to the sample management 114. At the sample management 114,the flow can be split into multiple flow paths 130, such as 3 flowpaths, where if desired a different sample can be injected into eachflow path and allowed to pass through one of the chromatographiccolumns, 116 a, 116 b and 116 c.

As another example, the flow can be split after the sample management114. A single flow can enter the sample management 114 and a commonsample can be injected into the single flow. Then, after the samplemanagement 114, the flow can be split into multiple paths to allow forparallel processing of the split flow by multiple chromatographiccolumns. For instance, a single flow with a common sample can begenerated in the sample management and then split into three flow pathsfor processing by the chromatographic columns 116 a, 116 b and 116 c.

In some embodiments, the flow management 101 of the chromatographicsystem 100 can be configured to allow the number of flow paths and thelocations where flow paths splits occur to be switched and controlled.The flow management 101 can include flow conduits that allow a number offlow paths to be simultaneously established. Further, the flowmanagement can include switching mechanisms, such as valves, that can beopened in closed at different locations that allow the number of flowpaths established at a particular location to be changed.

As an example, the flow management 101 can control flow conduits locatedwithin the sample management 114 that allow up to 3 flow paths to beestablished. The flow management can control branching mechanisms thatare located before and after the sample management. In a first mode, thebranching mechanism can be turned on prior to the flow reaching thesample management 114 to establish 3 separate flow paths (each flow pathcan be associated with a chromatographic column, such as 116 a, 116 band 116 c). Within each of the 3 flow paths, the sample management 114can inject a different sample which can then proceed into thechromatographic columns for analysis.

In a second mode, the flow management can be configured to switch off abranching mechanism prior to the sample management 114, such that only asingle flow path enters the sample management 114 and only a singlesample can be introduced. After the sample is injected into the singleflow path, a branching mechanism can be actuated that allows single flowpath to be split into multiple paths and pass through multiple columns.For instance, the single flow path can be split into three paths forprocessing by columns, 116 a, 116 b and 116 c. The chromatographicsystem can be configured to operate in the first mode or the secondmode.

The flow management 101 can be configured to control flow switchingmechanisms that allow different combinations of flow paths to becombined or to be split at various locations throughout thechromatographic system 100, such as within the solvent management,within the sample management 114, within the column management 117,within the detector management 120 or within the collection management124. For example, when possible three flow paths are available the flowmanagement 101 can be configured to control flow switching mechanismssuch that a single flow path, three separate flow paths or two separateflow paths can be generated at different times and at differentlocations within the chromatographic system 100. Further, at differentflow locations, the flow can be split from a single flow to two flows,from a single flow to three flows or from two flows to a single flow.

After the flow passes through each of the columns, it can be analyzedusing one or more different detectors 118. For instance, a flow cell canbe located near the end of the column that includes a transparent windowthat allows a light source to be shown through the flow cell. During thepassage of the column effluent (solvent mixture plus the physicallyseparated components of the original sample mixture) through the flowcell, light exiting the flow cell can be captured using a detector 118,such as a photomultiplier tube. As another example, after passingthrough a chromatographic column a portion of the flow can be divertedto an instrument, such as a mass spectrometer, for additional analysis.

In some embodiments, a single detector can be used for analysis ofmultiple flow paths. For instance, a single light source and singlephotomultiplier tube can be used to analyze flow flowing throughmultiple flow cells, such as three flow cells associated with each ofthe chromatographic columns, 116 a, 116 b and 116 c. An advantage ofinstrument sharing for multiple flow paths is reduced costs. Furtherdetails of instrument sharing are described with respect to FIGS.10A-10D.

At another point in the flow path, such as 136, the flow can exit therotatable element 132. The exit 136 from the rotatable element can occurwhile the rotatable element 132 is rotating or while the rotatableelement is stationary. As shown in the figure, after the flow leavesrotatable element 132, it can enter a waste or fraction collector. In128, the flow (elutes) entering the waste and/or fraction connectors canmark the end of the path.

Besides flow splitting, flow coalescing can also occur. In FIG. 1, anexample of flow coalescing is indicated where the multiple flowsconverge into a single flow. For example, when there is no samplecollection, all of the output from the chromatographic columns can becoalesced into a single flow that is diverted into a common wastecollector. Like the flow splitting, the chromatographic system 100 caninclude flow switching mechanisms that allow different flow paths to becoalesced at different locations where the locations where the flow iscoalesced can be varied from run to run. The flow management 101 can beconfigured to perform the flow control associated with the flowcoalescing, such as controlling the locations where flow coalescingoccurs.

Column Condition Management

One aspect of the chromatographic system 100 is an ability to establisha set of repeatable conditions within a chromatographic column. Thus,for a given flow path, system 100 can be configured to establish andmaintain a particular set of conditions associated with achromatographic column. The conditions can include but are not limitedto establishing and maintaining 1) a flow velocity within the column, 2)a solvent composition that varies with time during the chromatographicprocess, 3) a column temperature, 4) a solvent temperature and 5) arotational condition of the column, such as a constant angular velocity.

In particular embodiments, prior to introducing a sample into thechromatographic column, the chromatographic system 100 can be configuredto establish an initial steady-state condition, such as a steady flowvelocity, within the chromatographic column. Establishing an initialsteady-state condition can refer to determining that each of a selectedset of column parameters are varying within some acceptable range over aparticular time period. One reason for establishing steady-stateconditions prior to introduction of chromatographic sample is processrepeatability. Chromatographic experiments can be repeated for a numberof reasons and it can be desirable that the conditions of the experimentbe carried out each time in the same manner.

For example, chromatographic experiments can be repeated from thepurposes of fraction collection. For a given set of conditions within achromatographic column, a sample constituent can remain in the columnfor a certain period of time depending on its interactions within thechromatographic column. The amount of time that a sample constituentremains in the column can vary from constituent to constituent and is afunction of the column conditions (Chromatographic column conditions canbe intentionally selected to encourage a time differentiation between anamount of time one constituent remains in the column relative to anotherconstituent where the time differentiation is a reflection of thechromatographic separation efficiency of the column). Sampleconstituents exiting the column at a certain time can be collected. Theportion of the flow exiting the column at a certain time that iscollected can be referred to as a fraction. One advantage ofestablishing steady-state conditions in the chromatographic column priorto sample introduction is that a repeatable processes can be set-upwhere fractions are collected at a certain time after the sample isintroduced.

As another example, the chromatographic process can be used to determinea presence and an amount of a particular constituent in a sample. Thechromatographic process can be repeated a number of times to establishstatistical error bounds for a measurement, such as an amount of aconstituent in a sample. As a rule of thumb, a sampling error isproportional to 1/N^(1/2) (e.g., 100 samples are required to establish a10% error bar). Thus, the chromatographic process can be repeated manytimes on a particular sample to establish some reasonable error boundsfor a measurement, such as an amount of a particular sample constituent.One advantage of establishing steady-state conditions in thechromatographic column prior to sample introduction is to minimizeerrors associated with transient effects that can occur from run to run.

Referring to FIG. 1, a number of different components can be involved inestablishing steady-state flow conditions within column associated witha particular flow path. In one embodiment, this function can becontrolled by the flow management 101. To establish steady-stateconditions, the flow management 101 can receive data and sendinstructions to various components in the chromatographic system 100.

As an example, to establish a steady flow condition, the system 100 caninitialized for a run and then a rotatable element, such as 132, can bespun up via some angular velocity profile to a constant angularvelocity. During spin-up or after the constant velocity is reached, theflow management 101 can initiate flow on the rotatable element 132. Toinitiate the flow, the flow management can instruct a solvent deliverysystem, which can be located on or off the rotatable element 132 tobegin introducing a solvent into a flow path. The solvent can then beginflowing through the system 100, such as through a chromatographiccolumn, such as 116 a, 116 b or 116 c.

In one embodiment, to determine whether a steady-state mobile phasevelocity has been reached, the flow management 101 can send instructionsto the solvent management 108 to provide a solvent composition with afirst component that is varying in a known way, such as the firstcomponent increasing or decreasing as a fraction of the composition as afunction of time. The component that is being varied can be selectedsuch that it does not interact with the stationary phase of thechromatographic column and is detectable by one of the instruments inthe detector management 120. Using the information regarding how thefirst component is being varied and the information received from thedetector management 120, the flow management can determine a mobilephase velocity and its variation over time.

In another embodiment, a mass flow meter can be located in the flowpath, such as in the flow path after a flow cell. The mass flow metercan be used to determine a flow velocity. Based upon informationreceived from the mass flow meter, a system component, such as the flowmanagement 101, can determine whether a steady-state mobile phasevelocity has been achieved. When it is determined that the mobile phasevelocity as well as other column conditions are within acceptable valuesand their variation over time is within an acceptable limit, the columncan be identified as being ready for sample introduction.

As previously described, the rotatable element 132 can include a numberof columns, such as the three columns 116 a, 116 b and 116 c. Thedetermination regarding steady state flow and/or column conditions beingreached prior to sample introduction can be made on a column-by-columnbasis. The flow and/or column conditions determined for each column atleast prior to sample introduction can include but are not limited to amobile phase flow velocity, a solvent composition, a flow pressures(e.g., before and after the column), a flow temperatures (e.g., before,after the column), column temperatures (e.g., on the outside of thecolumn) and combinations thereof. As previously described, dataassociated with each column including whether steady state has beenreached for the column can be stored by one or more of the systemcomponents, such as the column management 117.

Measurements of flow and column conditions can also be made as afunction of time after a sample is introduced. For instance, a flowvelocity can be measured while a sample is progressing down achromatographic column. As another example, column temperatures on anoutside of the column at one or more locations along the column can berecorded as a sample is moving down a chromatographic column.

In some embodiments, the chromatographic system 100 can be configured todetermine whether flow and/or column conditions measured during achromatographic process are within acceptable ranges. A chromatographicprocess can take a particular amount of time and the column conditionscan be monitored during the time associated with the chromatographicprocess. An unacceptable condition can occur prior to a sample beingintroduced, such as a steady state condition not being reached, or aftersample introduction. For instance, for one or more of the columns, aparticular value at one time or a time variation of a parameter, such asa mobile phase flow velocity or a temperature can be out of anacceptable range. One or more column parameters being determined to beout of an acceptable range can result from a number of factors, such asa faulty flow cell associated with one of the columns (e.g., one of thewindows can be dirty), a faulty temperature sensor associated with oneof the columns, a faulty pressure sensor associated with one of thecolumns or a leak in one of the flow paths (a pressure sensor can beused to determined whether a leak has occurred).

Based upon determined flow and column conditions during a particular arun, the system 100 can be configured to identify acceptable andunacceptable columns. Certain columns can be identified as unacceptableprior to a sample being introduced. For instance, a column can beidentified as unacceptable when it is determined that steady stateconditions have not been met in a particular column. Other columns canbe identified as unacceptable after a sample is introduced. For example,an out of range pressure measurement can be detected for a particularcolumn while a sample is proceeding down a column and the columnassociated with the out of range pressure measurement can be identifiedas unacceptable.

In some embodiments, the system 100 can be configured to operate withsome of the columns identified as unacceptable where data collected fromunacceptable columns can be ignored and data gathered from columnsdetermined to be acceptable can be used. For example, a firstchromatographic column, such as 116 a, can be identified as unacceptablefor one of the following: 1) when initial steady-state conditions arenot established within an acceptable range, 2) a steady-state conditionis reached but a parameter is out of range or 3) a flow or a columncondition is determined to be out of an acceptable range while a sampleis proceeding down the chromatographic column. A second chromatographiccolumn, such as 116 b, can be identified as acceptable when it isdetermined that steady-state conditions are reached and all values arewithin an acceptable range prior to and after sample introduction, i.e.,during the chromatographic process associated with a particular run.Like the determination of whether steady-state conditions have beenestablished, the identification of columns as being acceptable orunacceptable for the purposes of using data gathered from the column canbe made on a column by column basis.

System Functions On or Off of a Rotatable Element

In various embodiments, functions of the chromatographic system 100 canbe performed by components located on a rotatable element, such as 132,or performed by components located off of a rotatable element. Whether aparticular function is performed on or off of rotatable element can varyfrom system to system and can also vary from run to run within a singlechromatographic system. This delineation of the system elements betweenrotatable elements and non-rotatable elements is provided forillustrative purposes only and is not limited to the example describedwith respect to FIG. 1.

In FIG. 1, a portion of the flow path and associated systems are shownon the rotatable element 132 and a portion of the flow path andassociated systems are shown located off of the rotatable element. Forexample, solvent management 108 and collection management 124 are shownoff the rotatable element 132 and the sample management 114, columnmanagement 117 and detector management 120 are shown located on therotatable element 132. In other embodiments, an entire chromatographicsystem 100 including solvent management 108, sample management 114,column management 117, detector management 120 and collection management124 can be located on rotatable elements, such as 132. Further, someparts of the chromatographic system 100 can be duplicated on rotatableand non-rotatable elements. For instance, a chromatographic system 100can include solvent management components, such as first solventreservoirs located on the rotatable element and a second solventreservoir located on a non-rotatable element. In some instances, thefirst solvent reservoir on the rotatable element 132 can be used. Inother instances, the second solvent reservoir on the off the rotatableelement 132 can be used. In yet other embodiments, the first solventreservoir on the rotatable element 132 and the second solvent reservoiroff the rotatable element can both be used.

In another example, the detector management 120 can include firstinstrumentation located on the rotatable element 132, such as a massspectrometer, and second instrumentation, such as a light source and aphotomultiplier tube located off of the rotatable element 132. Further,even when the light source and the photomultiplier tube are located offthe rotatable element, a flow cell that is needed to use thisinstrumentation can be located on the rotatable element 132. In someembodiments, only the first instrumentation located on the rotatableelement 132 can be used. In other embodiments, only the secondinstrumentation located off of the rotatable element can be used. In yetother embodiments, a combination of instruments located on and off ofthe rotatable element, such as the first instrumentation and the secondinstrumentation can be used.

Stationary-Rotational Interfaces

As described above, a chromatographic system can include one or morerotatable elements, such as 132. At various times, during the operationof the chromatographic system, one or more rotatable elements, such as132, can be at rest or can be rotating. When a rotatable element isrotating, it can be desirable to provide an interface that allows somequantity to be moved between a stationary element and the rotatableelement. Some examples of these quantities include fluids (e.g., gassesor liquids), power and data. A single rotational element can be coupledto stationary elements via multiple stationary-rotational interfaces.For instance, a rotatable element can coupled to a number of differentstationary elements via multiple fluid interfaces, power interfaces anddata interfaces.

In particular embodiments, an interface can be designed to move aquantity in only one direction, such as from a stationary element to arotating element or from a rotating element to a stationary element. Forinstance, a first fluid interface can be configured to only deliverfluid from a stationary element to a rotating element while a secondfluid interface can be used to only deliver fluid from a rotatableelement to a stationary element. In other embodiments, an interface canbe configured as bi-directional and allow a quantity to be both movedfrom a stationary element to rotatable element and from the rotatableelement to the stationary element simultaneously or at different times.

For instance, a single fluid interface can be configured to deliverfluid from a stationary element to a rotatable element and to receivefluid from a rotating element and deliver it to a stationary element atthe same time. In this example the flow can be moving between thestationary and rotatable elements via separate flow conduits. In anotherexample, at first time a single fluid conduit can be used to deliverfluid from the stationary element to a rotatable element and at a secondtime, it can be used to receive fluid from the rotatable element anddeliver it to a stationary element.

Operational Modes

The chromatographic systems described herein, such as system 100 in FIG.1, can be configured to operate a number of different modes. During thedifferent operational modes, the chromatographic system can performdifferent functions. A few examples of different operation modes areinitialization, operational, spin-up, spin down, chromatographicprocessing and data collection between runs, and malfunction.

During initialization, the chromatographic system can perform a numberself-checks to determine a status of its various system components, suchas but not limited to instrumentation statuses, fluid reservoir levelstatuses, gas pressure statuses, pump statuses, valve statuses, a motorstatus, balancing checks, device power statuses, and devicecommunication statuses. These self checks can be associated differentsystem elements, such as flow management 101, solvent management 108,sample management 114, data management 115, column management 117,detector management 120, collection management 124 and rotor management140. After self-checks have been performed, it can be determined whetherthe chromatographic system can enter into an operational mode or whethercorrective actions are needed for one or more of chromatographic systemcomponents. The chromatographic system can output whether correctiveactions are needed or chromatographic system can indicate that it is inan operational state.

After the chromatographic system enters into an operational state, itcan be configured for a particular run. In some embodiments, thechromatographic system, such as via system management 103, can providean interface that allows a user to specify one or more adjustableparameters of the run. After parameter of a particular run is specified,the chromatographic system can implement the run using the selectedparameters. For example the chromatographic system can spin up one ormore rotatable elements, such as element 132, from a resting state tosome angular velocity target according to some angular velocity profile.Spin-up can include rotor management functions, such as checking andadjusting a balance of the system and flow management functions, such asinitial fluid introduction into the system and leak detection.

After spin-up, the system can determine whether it has reachedconditions, such as a steady flow condition, such that chromatographicprocessing can begin. When it is ready for chromatographic processing,functions such as sample introduction and data collection can begin. Thesystem can monitor the chromatographic processing until it determines arun is completed. When a run is completed, the chromatographic systemcan begin a spin-down procedure.

During spin-down, a rotatable element 132 can be decelerated accordingto some angular velocity profile from a particular angular velocity torest. Prior to or during spin down, the flow management 101 can changeflow functions, such as shutting down the flow in the rotatable element132. After spin-down, while the rotor is at rest, functions, such asdraining on-rotor flow reservoirs or removing fractions collected on therotatable element can be performed. Between runs, functions such asflushing fluids from the columns in the rotatable element for thepurposes of cleaning can be performed. Flushing the columns can involvea spin-up and spin-down of the rotatable element, such as 132, to allowfluid to be pushed through system.

A malfunction can occur during any of the operation modes. When amalfunction is detected, the system 100 can go into a unique malfunctionmode depending on the type of malfunction. For instance, if a leak isdetected during spin-up or a sudden change in balance is detected whilethe rotatable element is rotating, such as a result of a leak or acomponent failure, a spin-down malfunction mode can be implemented. Thespin-down malfunction mode can involve de-accelerating the rotatableelement faster than it is normally de-accelerated during normaloperations.

Rotatable Elements

In FIGS. 2-8, details of one embodiment of a rotatable element that canbe utilized with chromatographic system are described. Additionalembodiments of a rotatable element are described with respect to FIGS.9A-9C. In particular, with respect to FIGS. 2 and 3, a rotor assemblyincluding a number of chromatographic columns is described. With respectto FIGS. 4A and 4B and 6 a number of components of the rotor assemblyare discussed. In FIGS. 5A-5C, further details of the rotor assembly aredescribed including a discussion of a flow path through the rotorassembly. In FIGS. 7A-C and 8, a number of components for allowing therotor assembly to be utilized as part of a chromatographic system arediscussed. With respect to FIGS. 9A, 9B and 9C, embodiments of rotorassemblies including sample injection, column conditioning, powergeneration, off-rotor communications, fraction collection and a reactionchamber are described. With respect to FIGS. 10A-10D, an alternatesystem for performing centrifugal liquid chromatography thatincorporates a swinging bucket design is described.

FIGS. 2 and 3 are a side view and top view of an embodiment of a rotorassembly 200 configured for use in a chromatographic system, such as thechromatographic system described with respect to FIG. 1. The rotorassembly is one embodiment of a rotatable element. As is described withrespect to FIG. 1, the components on or off of a rotatable element canvary from chromatographic system to chromatographic system. Thus, theembodiments described with respect to FIGS. 2-9C are provided for thepurposes of illustration only.

The rotor assembly 200 can include a platter assembly 202, a manifoldassembly 204, an adapter plate assembly 214 and a liquid introductionassembly including a mixing chamber 228 located in a center portion ofthe platter assembly 202. The manifold assembly 204 includes a diskportion 206 and a column portion 208. The liquid introduction assemblycan be coupled to the platter 202 via a number of fasteners. The platter202 can be coupled with fasteners to the disk portion 206 of themanifold assembly. A bottom of the column portion 208 of the manifoldassembly can be coupled to the adapter plate assembly 214 via an adapterplate (see FIG. 6 for more details of the adapter plate assembly 214 andthe adapter plate).

A reservoir 210 is coupled to the adapter plate assembly 214. Theadapter plate assembly includes an interface to a shaft, such as taperedshaft 212. The shaft provides a coupling mechanism to a motor (notshown) that allows an angular velocity to be imparted to the rotorassembly 200. The angular velocity of the rotor assembly can be variedas a function of time based upon operating commands sent to the motor.In one embodiment, the motor can be a Sorvall Model RC5C (ThermoScientific, Waltham, Mass.), which can provide RPM's between 300 to22,000 and RCF's up to 55,000 g's. Further details of the manifoldassembly 204 and the adapter plate assembly 214 including a coupling ofthese components is illustrated with respect to FIGS. 5A and 6. Ingeneral, any suitable mechanism can be used to drive a rotor and impartan angular velocity to the rotor. For instance, systems using acompressed gas, such as compressed air, can be used to drive a rotor.

As is illustrated in FIG. 3, the platter 202 includes a number ofcolumns, such as 230. The columns, which can also be referred to aschromatographic enclosures, include a hollow interior portion in whichmaterials, such as materials used to perform a chromatographic process,can be packed. During operation, the hollow interior portion can providea flow conduit for a flow path (e.g., see FIG. 5A). When packed withchromatographic materials, a chromatographic separation can occur alonga portion of flow path within the columns, such as 230.

In the embodiment shown in FIG. 3, 24 columns are arranged around theplatter 202. In some embodiments, as is shown, the columns, such as 230,can include a common length, a common outer diameter and a common innerdiameter. The columns can also be generated from a common material, suchas a metal or metal alloy. Further, the columns can be distributedaround the circumference of the platter 202, such that an equal angularspacing is provided between each of the columns. In yet otherembodiments, some flow conduits used in the rotor assembly can begenerated from a flexible material, such as a flexible plastic.

In other embodiments, the number of columns on a platter can be varied,such that a platter 202 can include more or less than the 24 columnsthat are shown. In addition, on a single platter, a length of thecolumn, an outer diameter and an inner diameter of the column can varyfrom column to column over the number of columns arranged on platter202. Further, a composition of a chromatographic material, such as astationary phase material, packed within the column can vary from columnto column. Also, the angular spacing between columns does not have to beequal and the spacing between columns can vary from column to column.Further, the material form which the column is generated can vary fromcolumn to column (e.g., first column can be composed of a ceramicmaterial and a second column can be composed of a metal alloy.)

In yet other embodiments, a rotor assembly can include a number ofplatters. The platters can be arranged in a stacked configuration one ontop of the other. In one embodiment, a transmission and/or mechanismscan be associated with each platter that can be used to allow theplatters to engage and disengage from a rotating shaft and to rotate atdifferent speeds from one another. From platter to platter, the numberof columns can be varied. Further, the column parameters, describedabove, such as the column length, outer diameter, inner diameter, columnmaterial, column spacing and column packing material can vary fromplatter to platter.

In the embodiment in FIG. 3, a flow cell, such as 232 can be located atthe end of each column. The flow cell 232 can include windows and ahollow interior portion. The hollow interior portion can provide a flowconduit for flow that exits an associated column, such as a column inwhich a chromatographic process is performed. The windows can allow alight source to be passed through the flow cell.

In some embodiments, the flow cells located at the end of each columncan be a common flow cell with a similar design, such as windows locatedon a top and a bottom portion of the flow cell and common dimensions forthe interior portion. In other embodiments, the flow cell associatedwith each column can vary from column to column. For instance, a firstcolumn on the platter can be associated with a flow cell that haswindows located on its sides while a second column can be associatedwith a flow cell that has windows located on the top and bottom. Inaddition, if column parameters vary from column to column, such as acolumn diameter, then the flow cell parameters, such as a size of aninterior portion of the flow conduit, can also vary.

In other embodiments, a flow cell does not have to be associated witheach column. For instance, a first column can be associated with a flowcell while a second column can be associated with a mass spectrometerand not even be associated with a flow cell. In another example, a flowcell and a mass spectrometer can be associated with a column. Thus, ingeneral, the instrumentation associated with a chromatographic columncan vary from column to column on a single platter and can also varybetween platters. Further details of instrumentation that can beutilized with a column, such as a column in which a chromatographicprocess can be performed, are described below in the section tilted,“Instrumentation.”

Returning to FIGS. 2 and 3, the columns, such as 230, can be held inplace via a column clamp ring 216. The column clamp ring 216 can beattached to the platter 202 via a plurality of fasteners, such as 236.In one embodiment, a load associated with each column can be channeledthrough a load support mechanism, such as a pre-load nut 234. Thepre-load nut 234 is visible through the column clamp ring 216. Thepre-load nut can be coupled to platter 202.

During rotation, the columns experience a large RCF, which can behundred's to thousands of g's. When a flow cell is located at the end ofa column near an outer edge of the platter 202, as is shown in FIG. 3,the large RCF experienced by the column can be transferred as a force tothe flow cell, such as 232. If too large a force is transferred to theflow cell, a shape of the flow cell can be distorted including a shapeof the flow cell windows, such as 242. A distortion in the flow cell,such as a distortion in the windows, can cause degradation in theoptical properties of the flow cell and hence can degrade a measurementquality associated with using the flow cell. A load support mechanism,such as a pre-load nut, can be used to bear some of the loads generatedby the column during rotation and hence, reduce potential loads on theflow cells, such as 232. The pre-load nut associated with each columncan be mechanically coupled to the platter 202 such that a portion of aload on the column is transferred to the platter or some other supportstructure rather than to the flow cell.

The liquid introduction assembly including a mixing chamber 228 can belocated in a center 238 a of the rotor assembly 200. In one embodiment,fluid can enter the mixing chamber 228 from a fluid source that remainsstationary while the rotor assembly 200 is rotating. For instance, oneor more flow conduits can be inserted down into the mixing chamber 228where the conduits can remain stationary while the mixing chamber 228 isrotating. During operation, fluid can exit the conduits and enter themixing chamber 228 to provide a continuous and controlled supply offluid to the rotor assembly 200.

An assembly including a gas bearing where the gas bearing provides aninterface between the assembly including the conduits and the rotorassembly 200 can support the conduits. The rotor assembly 200 caninclude a seat 240 for the gas bearing. While the rotor assembly isrotating, the gas bearing can rest on the seat 240. Further details ofthe gas bearing support assembly that can include the stationary fluidsource and support for the conduits are described with respect to FIGS.7A-7C.

Fluid can enter the mixing chamber 228 and then move along a flow pathfrom the center 238 a of the platter 202 to an edge of the platter 238b. In some embodiments, the columns can be arranged proximately alongradial lines (i.e., lines that pass through a center of the platter 202)such that a direction of the bulk flow is along one of the radial lines.In other embodiments, one or more of the columns can be arranged along anon-radial line that does not pass through a center of the platter.Nevertheless, a centrifugal force component along the non-line radialline can still move the flow through the non-radially aligned column.

In one embodiment, fluid components from one or more sources can enterthe mixing chamber 228 and can be mixed. Mixing can be generated from arotation of mixing pins in the mixing chamber. The mixed fluidcomponents can exit the mixing chamber 228 through a number of ports,such as a port associated with each column. Thus, in one embodiment, twoor more of the columns can receive fluid from a common source. Moredetails of the mixing chamber 228 are described with respect to FIG. 5A.

A fluid mixture can then move through each of the columns and into anassociated flow cell near the edge 238 b of the platter 202. Afterpassing through the flow cell, such as 232, the fluid can move beyondthe edge 238 b of the platter 202 and into a return segment link 220.The return segment links, such as 220, can include a flow channel thatroutes the fluid exiting a flow cell along the edge of the platter 202.The return segment link, such as 220, can be connected to a return flowchannel that is next to a column that feeds it fluid. Once the flowenters the return channel, it can be moving generally away from edge 238b and towards center 238 a.

In this embodiment, a fluid flow can enter a beginning of a column thatis located near a center of the platter 202 and exit an end of columnnear the platter edge. The column can include a chromatographic packingmaterial such that a chromatographic separation of components in a fluidmixture can occur as the flow moves from the beginning of the column tothe end of the column. The centrifugal forces on the fluid in the columncan increase as fluid in the column moves down the column toward theedge of the platter 202. For this orientation, the increasingcentrifugal forces can result in a fluid pressure increasing along alength of the column as the chromatographic process occurs. Thispressure profile along the column differs from chromatographic systems,such as a high performance liquid chromatography (HPLC). In HPLC,pressure is used to move fluid from a beginning of the chromatographiccolumn to an end of the chromatographic column. Thus, the pressure dropsfrom the beginning of the column to the end of the column as opposed toincreasing.

In particular embodiments, a maximum operational pressure of the rotorassembly can be about 100 PSI. For instance, the peak pressure as thepressure increases from a center of the rotor to the edge of the rotorcan be less than 100 PSI. In other embodiments, the maximum operationalpressure can be less than 50 PSI. The maximum pressure levels, such asbelow 100 PSI, are much less than HPLC maximum pressure levels, whichcan be 1000's of PSI for smaller particles sizes.

In a particular embodiment, the return segment links, such as 220, canbe linked together via connectors, such as 241. The inner surface ofeach return segment link can conform to an outer surface of the platter202. For instance, when the outer surface of the platter 202 is curved,then the links can conform to the curvature of the outer surface of theplatter 202. The links can each be coupled to the platter via fasteners,such as 222. Further details, of the return segment links are discussedwith respect to FIGS. 5A-5C.

After moving through the return segment link and into a return flowchannel in platter 202, the flow can move into a circumferentiallyaligned channel in the disk portion 206 of manifold assembly 204. Thecircumferentially aligned channel can drain into the manifold blocks,such as 225. Each manifold block can be coupled to an adjustable tube226, which is coupled to the column portion 208 of manifold assembly vialock nut 224. The flow can then move through the column portion of themanifold assembly and eventually into reservoir 210. Fluid can beperiodically drained from the reservoir 210 via the reservoir drainagetubes, 218 a and 218 b. More details of the flow path including the flowpath through the interior of the rotor assembly 200 are described withrespect to FIGS. 5A-5B

FIGS. 4A and 4B are a top perspective view and a bottom view of manifoldassembly 204. The disk portion of the manifold assembly can include acircumferentially aligned drainage channel 248. An outer 242 a and innerchannel 242 b can surround the drainage channel 248. The outer and innerchannels, 242 a and 242 b, can support circular gaskets (not shown).When the top of manifold assembly is coupled to the bottom of theplatter 202, the circular gaskets can press against the bottom of theplatter 202 and prevent fluids from leaking from the drainage channel248 during operation of the rotor assembly 200.

The drainage channel 248 can include a number of drainage holes, such as246. Six drainage holes are shown in the FIG. 4A. The drainage holes canbe each coupled to a manifold block, such as 225. The manifold diskportion 206 can include a number of apertures, such as 251, forfasteners, such as 254, that couple the manifold blocks, such as 225, tothe disk portion.

An adjustable tube can be coupled to each manifold block 225. Anaperture 252 in the column portion 208 of the manifold assembly canallow an end portion 250 of the adjustable tube to be inserted throughthe column 208 and to drain into a central hollow portion of the column208. In FIG. 4B, end portions, such as 250, of adjustable tubes, such as225, can be seen within the central drainage column 244 of the manifoldcolumn 208.

The manifold assembly 204 can be coupled to the adapter plate assembly214 which includes an adapter plate (see FIG. 6) via mounting holes,such as 258, in the bottom ring portion 256 of the manifold assembly204. Fasteners can be inserted through the mounting holes to couple themanifold assembly 204 to the adapter plate assembly 214. A bottomopening 245 in drainage column 244 can be coupled to drainage channelsin the adapter plate assembly 214 to allow fluid to exit the drainagecolumn 244 and travel through the adapter plate assembly into thereservoir 210.

The reservoir 210 can include a number of drainage tubes, such as 218 aand 218 b in FIG. 2. As previously described, reservoir 210 can beperiodically drained via the drainage tubes. In one embodiment, thedrainage tubes can pass through the bottom of the disk portion 206 ofthe manifold assembly 204 via apertures, such as 255. The platter 202can also include apertures (not shown) that allow the drainage tubes topass through the platter.

As is described with respect to FIG. 1, in embodiments described herein,flow paths can be split and then coalesced. As an example, in the rotorassembly 200, a single flow path starts in the mixing chamber 228 andthen is split into 24 separate flow paths (see FIGS. 2 and 3). As isshown in FIGS. 4A and 4B, the 24 separate flow paths can coalesce into asingle drainage channel 248. The single drainage channel 248 can splitinto 6 separate flow paths through the manifold blocks, such as 225,which then are coalesced into a common drainage column 244.

Referring to FIG. 4B, the diameter of the disk portion 206 of themanifold assembly is smaller than the diameter of platter 202. Thesmaller diameter of the disk portion 206 allows the flow windows on thebottom of the flow cells, such as 232 a and 232 b, to be accessed. Thus,if a light source is provided proximate to the top portion of the flowcells, light emitted from the light source that has passed through theflow cell and exited via the bottom window can be gathered. Aninstrument mount for providing a light source and gathering lightexiting from a flow cell while the rotor assembly is rotating isdescribed with respect to FIG. 8.

In FIG. 4B, the platter 202 is rendered as partially transparent. Thus,a portion of the return flow channel tubes associated with each flowcell can be viewed. For instance, return flow channel conduits, 252 aand 252 b, which are each next to flow cells, 232 a and 232 b,respectively are visible. Fasteners, such as 222, are also visible. Thefasteners, such as 222, couple the return segment links to the platter202.

In one embodiment, the return flow channel conduits can be formed bydrilling into a solid portion of the platter 202. As previouslydescribed, the return flow channels, such as 252 a and 252 b, can routefluid that has exited the flow cells and entered into the return segmentlinks to move towards the circumferentially aligned drainage channel 248in the disk portion of the manifold assembly 204 and away from the edgeof the platter 202.

The return flow channel can be positioned to the side of each flow cell(as opposed to below the cell) to allow access to the bottom windows ofthe flow cells. In other embodiments, such as to accommodate differentinstrumentation, the return flow channels can be routed along adifferent path. Further details of a return flow channels are describedwith respect to FIG. 5B.

FIG. 5A is a side cross sectional view of a rotor assembly 200 with anillustrated flow path. The flow path can start in solvent reservoirs 285where one or more solvents can be delivered to the flow path. In someembodiments, a number of different solvents can be simultaneouslydelivered to the flow path where the fractions of solvent components inthe solvent composition can vary over time. In this embodiment, thesolvent reservoirs are not located on the rotor assembly 200. Aspreviously described, in other embodiments, one or more solventreservoirs can be located on the rotor assembly 200. For instance, asolvent reservoir can be associated with each or a portion of columns,such as 230 a and 230 b, on the platter 202. The one or more solventscan be delivered by a pump (not shown). An embodiment of a solventreservoir located on a rotor assembly is described in more detail withrespect to FIGS. 10A-10D.

Returning to FIG. 5A, as previously described with respect to FIGS. 2and 3, a gas bearing support assembly can include or more flow conduits(see FIGS. 7A-7C and 8 for more details of the gas bearing supportassembly 400). The one or more flow conduits can be part of a flow path,such as 260. The flow conduits in the gas bearing support assembly 400can be used to deliver fluids, such as solvents to the mixing chamber228. In one embodiment, a flow conduit in the gas bearing supportassembly 400 can also be used to deliver a sample for use in achromatographic process to the mixing chamber 228. In some instances,the sample can be mixed with one or more solvents in the mixing chamber228.

In the mixing chamber 228, a number of mixing pins, such as 275, can mixone or more fluids entering the mixing chamber. The mixing pins canrotate according to the angular velocity of the rotor assembly 200. Attypical rotational speeds, the mixing pins can tend to remove air fromthe fluid. Thus, when only a single fluid is delivered to the mixingchamber and a mixture of fluids is not created, an advantage of themixing chamber 228 can be removing air from the single fluid prior toits entry into the columns, such as 230 a and 230 b.

The mixing chamber 228 includes a number of apertures, such as 276, thatallow fluid to exit from the mixing chamber. In one embodiment, eachaperture is in fluid communication with a chromatographic column wherethe column contains a single chromatographic stationary phase. In otherembodiments, one aperture, such as 276, in the mixing chamber 228 can bein fluid communications with multiple chromatographic columns. Forinstance, in a tube-within-tube a tube design, a number of tubes can beplaced within the interior of a hollow cylinder where each tube containsa separate chromatographic stationary phase. An entrance to each of thetubes can be provided near a top of the hollow cylinder that is in fluidcommunication with one of the apertures in the mixing chamber. Duringoperation, fluid exits the mixing chamber via the apertures and thenenters each of the tubes. The tubes can have circular or non-circularcross sections.

In another embodiment, a valve can be situated proximate to eachaperture. The valves can be used to control a rate of fluid enteringeach aperture. For instance, the valves can be used to control a size ofan opening associated with each aperture. In some instances, the valvescan close to prevent fluid from exiting via a particular aperture. Forexample, if a leak is detected downstream of a first aperture, the valveassociated with the first aperture can be actuated to prevent additionalfluid from exiting via the first aperture.

A number of columns, such as 230 a and 230 b, can extend from the mixingchamber to the edge of the platter 202. In one embodiment, the columnscan be arranged in symmetric pairs to help balance the rotor assembly200 when it rotates. For instance, columns 230 a and 230 b can be asymmetric pair of columns with similar mass properties. However, aspreviously described, the mass properties of column pairs can vary fromcolumn pair to column pair.

In a particular embodiment, the columns can extend along a line that isperpendicular to the axis of rotation 265 through the center of therotor assembly 200. In other embodiments, the columns can extend along aline that is not perpendicular to the axis of rotation (e.g., two anglescan be defined relative to the axis of rotation that determines the linethat each column follows such as two angles used in a sphericalcoordinate system). For instance, each of the columns can be slopeddownward or upward from the center axis 265 of the rotor assembly to theedge of the platter 202. The platter 202 can be thicker or includeadditional support structure to support columns that extend above orbelow the platter 202.

As another example, rather than a single column 230 a extending towardthe edge of the rotor, the platter 202 can include two columns at thislocation one sloped downward and one sloped upwards that connect to themixing chamber and lead to the edge of the platter 202. In yet anotherexample, the platter 202 can include 3 columns, column 230 a which isstraight across the platter, a second column sloped upward above column230 a and a third column sloped downward below column 230 a. Each of thecolumns can be connected to the mixing chamber 228 or can be connectedto separate fluid reservoirs.

In yet other embodiments, an inner area through which fluid flows in theflow conduits between the mixing chamber 228 and the edge of the platter202 can be constant. For instance, the inner area of the column 230 aand the flow cell 232 can be approximately constant. In one embodiment,such as inside the flow cell 232 or column 230 a, the inner area of flowconduit can remain constant but a shape of the perimeter of the innerarea can change along the flow conduit. For instance, the shape of theinner area of the flow conduit can transition from a square of with anarea to a circle of the same area where during the transition betweenthe two shapes the area remains constant.

In other embodiments, the inner area of the flow conduits between themixing chamber and the edge of the platter 202 can vary between themixing chamber and the edge of the platter 202. For instance, a flowrestrictor with a small inner area than the rest of the conduits can beplaced near the end of column 230 a or after flow cell 232 to slow downthe flow velocity.

During operation, the rotation of the rotor assembly 200 can cause fluidto build up on side walls 266 of the mixing chamber 228. In oneembodiment, a portion of the side walls 266 can be generally parallel toan axis of rotation of the rotor assembly 200 while other portions canbe angled relative to the center axis of rotation. Other mixing chambersshapes are possible. For instance, a lower portion of the mixing chamber228 can be a bowl shaped to some curvature profile.

When a fluid is added to the mixing chamber 228 while the rotor assembly200 is rotating, a fluid head of some thickness 268 can build up on theside walls of the mixing chamber 228. Thus, boundaries of the fluid headcan include a portion of the top, bottom and side walls of the mixingchamber and a free boundary that extends some distance (e.g., the fluidhead thickness) into the mixing chamber 228. The free boundary can beapproximately described as a cylindrical shaped wall of fluid that isparallel to an axis rotation of the rotor assembly 200.

As shown in FIG. 3, a top of the mixing chamber 228 can be partiallycovered but can include an aperture. In one embodiment, the aperture canbe circular but other shapes can also be used. As described above, oneor more flow conduits can be passed through the aperture to allow fluidto be delivered to the mixing chamber 228. As is described in moredetail with respect to FIGS. 7A-7C, the top of the mixing chamber can becovered by a gas bearing.

The gas bearing can enclose the mixing chamber and can prevent fluid andfluid vapors contained in the mixing chamber 228 from escaping. The gasbearing can remain stationary while the rotor assembly 200 including themixing chamber 228 is rotating. Thus, during operation, the mixingchamber 228 enclosure can be formed from a portion of components thatcan be rotating and a portion of the components that can benon-rotating. In other embodiments, an enclosure can be form formedbetween two components rotating at different rates. For example, the gasbearing that sits on top of the mixing chamber can be associated with acomponent that can remain stationary or rotate at a different rate thanthe rotor assembly.

In addition, a fluid enclosure including stationary and non-rotatingcomponents or components rotating at different rates is not limited touse as a mixing chamber. For instance, the mixing pins can be removedfrom the mixing chamber to form a fluid enclosure that is used as afluid reservoir. Also, a similar enclosure can be used to drain fluidfrom the rotor assembly. In this example, a bottom portion of the rotorassembly 200 can rest on a gas bearing. The gas bearing can bestationary and include a chamber that is topped by the rotor assembly200. A flow conduit that rotates with the rotor assembly 200 can extendfrom the rotor assembly 200 into the chamber to deliver fluid into thechamber while the rotor assembly is rotating. Then, fluid can then beextracted from the chamber.

As is described in more detail as follows with respect to FIG. 5A andFIGS. 5B, 5C, 7A, 7B and 7C. Along a flow path on the rotor assembly 200that starts in the mixing chamber 228 and ends at some location, such asin the reservoir 210 or via an interface that allows the flow to leavethe rotor assembly 200, the flow can be contained. One advantage ofcontaining the flow in various enclosures along its path through therotor assembly is safety. Many fluids that can be utilized with thedevices described herein can be dangerous. For example, some fluid canbe flammable while other fluids can be health damaging, such as being acancer causing agent in humans. Thus, keeping the fluids contained inrotor assembly 200 along its flow path can provide for safer operatingconditions than including open air interfaces on the rotor assembly 200where fluids or fluid vapor can more easily escape from the rotorassembly 200 (If the fluids used with the rotor are not unsafe, than therotor assembly can be designed that includes interfaces where the fluidis not totally contained, such as an open air interfaces where fluidmoves through a space that is at least partially unenclosed).

Returning to FIG. 5A, the rotor assembly 200 can be configured such thata thickness of the fluid head 268 in the mixing chamber can be adjusted.For instance, thickness of the fluid head can be adjusted by changingthe geometry of the flow path in the rotor assembly 200 so that thefluid head does not extend into an inner perimeter of the aperture inthe top of the mixing chamber. To illustrate how the fluid headthickness 268 can be adjusted, first a flow path through the mixingchamber 228 to the exit 250 of the adjustable tube 226 is describedincluding an initialization of the flow along this flow path. Second, anequilibrium condition is described where forces acting on the fluid intwo segments of the described flow path can balance. Finally,adjustments to the configuration of the rotor assembly 200 that canaffect the equilibrium condition including the fluid head thickness 268are discussed. At the equilibrium condition, the fluid head thickness268 in the mixing chamber can be a certain value. Thus, changing theequilibrium condition can change the fluid head thickness in the mixingchamber 228.

In the rotor assembly 200, a line 262 is drawn at a constant radius 264from a center axis 265 of the rotor assembly. The flow path 260 is shownnear the center axis 265 of the rotor assembly. The line 262 crosses theflow path through the rotor assembly 200 in the mixing chamber 228 andnear the exit 250 of the adjustable tube 226. The exit 250 can drainfluid from the adjustable tube into to central drainage column 244 ofthe manifold assembly 204.

The rotor assembly 200 can rotate with an angular velocity about itscenter axis 265 when a force is imparted to the rotor assembly by amotor (not shown). As previously described, the centrifugal forcesincrease as a distance from the center axis of rotation 265 increasesand the angular velocity of the rotor assembly increases. Thecentrifugal forces can move fluid through the rotor assembly 200.

For the purposes of discussion, two segments of the flow path throughthe rotor assembly 200 can be defined. A first flow path segment can bedefined as starting at the wall of fluid on free boundary of the fluidhead 268 in the mixing chamber 228 and moving outwards (increasingradius) to end within the return link 220. A second flow path segmentcan be defined as starting at the exit 250 and moving outwards(increasing radius) to end within the return segment link 220. The firstand second flow path segments can be joined in the return segment link220.

In one embodiment, to initialize the flow in the two flow segments, therotor can be spun up from rest to a constant angular velocity. Fluidintroduction can begin during spin up (prior to reaching the targetangular velocity) or after the rotor assembly 200 has reached the targetangular velocity. In some embodiments, residual fluid can remain in therotor assembly from a previous run. The residual fluid can fill all orportions of the first and second flow path segments. Thus, a portion ofthe first and second flow path segments can be dry at initializationwhile other portions can include residual fluid.

During spin-up, fluid introduction can begin in the mixing chamber 228.Not all rotor assemblies 200 necessarily include a mixing chamber 228.In some embodiments, a rotor assembly 200 can have multiple fluidintroduction points, such as separate introduction points for eachcolumn such that a common introduction point, such as 228, is not used.Thus, this example is provided for the purposes of illustration only.

Typically, for chromatographic separation process performed in thecolumns, such as 230 a and 230 b, fluid can be passed from one in end ofthe column to the other end of the column. In the embodiment in FIG. 5A,fluid can be introduced at the beginning of the column 270 that isclosest to the mixing chamber 228 and flow through the column to an end280. To get flow to the beginning of the column 270, various flow pathscan be configured. For instance, a flow path is shown in FIG. 5A whereflow starts near the center of the rotor in the mixing chamber and movescontinually away from the center on its way to the beginning of thecolumn. In other embodiments, the flow can start at a radius than isgreater than the radius of the beginning of the column and then movetowards the center of the rotor until it reaches the beginning of thecolumn 270 and reverses direction to flow into the beginning of thecolumn 279.

In yet other embodiments, chromatographic columns, such as 230 a and 230b, can be located on a return flow segment. First, the flow can movefrom a center 265 of the rotor assembly 200. At some radial distancefrom the center of the rotor back it can be turned towards the center ofthe rotor to enter a return flow segment. The chromatographic column canbe located on the portion of the flow path where the flow is movingtowards the center of the rotor, i.e., the return flow segment. Forexample, a flow path can be configured where the flow moves from the end280 of the column 230 a to the beginning of the column 270 while achromatographic separation process is performed in the column 230 a. Inthis example, instrumentation, such as a flow cell or a massspectrometer can be located at the beginning 270 of the column 230 a,i.e., the instrumentation configuration can reverse of what is shown inFIG. 5A.

In other embodiments, a chromatographic column does not have to bestraight. The column can be curved in some manner from the center of therotor to the edge of the rotor. In general, a flow conduit that followssome path in the rotor assembly 200 can be used to perform achromatographic process. The chromatographic flow conduit can bestraight or curved. In the chromatographic flow conduit, flow can begenerally moving towards the center of the rotor assembly or away fromthe center of the rotor assembly 200. Prior to reaching a beginning ofthe chromatographic flow conduit where the flow enters, the flow can bemoving along a flow path where the radial distance from the center ofthe rotor assembly is varying including where the radial distance isincreasing, decreasing, constant or a combinations thereof along theflow path. Further, the flow can be moving at radial distances that aregreater or lesser than the radial distance where the flow enters thechromatographic flow conduit. Finally, after reaching an end of thechromatographic flow conduit and exiting, the flow path can be movingalong a flow path where the radial distance from the center of the rotorassembly is varying including where the radial distance is increasing,decreasing, constant or combinations thereof along the flow path.

During different modes of operation of the rotor assembly 200, reverseflow configurations can also be achieved. For instance, if the flow isblocked from reaching exit 250, such as via actuation of a valve, on thereturn flow path segment from the edge of the platter 202 towards thecenter 265, then fluid that is at a radial distance above the valvelocation can begin flowing back towards the edge of the platter 202 andthen reverse directions and flow back towards the center 265 of therotor assembly 200 via the flow cell 232 and the column 230 a. Thus, inparticular embodiments, flow paths can be configured where the flow canmove in one direction at some times and in an opposite direction othertimes.

Returning to FIG. 5A, during flow initialization, the fluid introducedinto the mixing chamber 228 can exit the mixing chamber 228 and canenter into the columns, such as 230 a and 230 b. Each column can includetwo detachable caps, such as, 272 and 274, which are shown coupled tocolumn 230 b. In one embodiment, the two detachable caps can be threadedand screwed onto the ends of each column. The caps can each include aflow conduit. In particular embodiments, the flow conduit in eachdetachable cap is the same area as flow conduit in its associatedcolumn.

The first attachable cap, such as 272, can provide an interface for thecolumn to the liquid introduction assembly including the mixing chamber228. In some embodiments, the first attachable cap can include asurface, such as a seat or a groove, on which a gasket of some type isplaced. The gasket can be used to form a seal between the mixing chamberassembly and the first attachable cap. Similarly, the second attachablecap, such as 274, can provide an interface between each column and anassociated flow cell, such as 232. The second attachable cap can includesurfaces for interfacing with the flow cell, such as protuberance thatis inserted into the flow cell 232 as well as a seat or grooves forholding one or more sealing mechanisms, such as a gasket.

If fluid is first introduced into the mixing chamber during spin-up,then the rotor assembly 200 may have to reach an angular velocitythreshold before fluid can enter into the columns, such as 230. When theangular velocity threshold is reached, the fluid in the mixing chamber228 can enter into the column 230 a and progress down from a beginning270 of the column to an end 280 of the column to establish a flow path278 within the column 230 a. The angular velocity threshold can dependon a chromatographic packing material property, such as a size of thepacking material particles, used in the chromatographic column 230.Typically, smaller diameter packing particles can require a greater RCFto move the flow through the packing material as compared to largerdiameter particles. Since RCF is proportional to the angular velocitysquared, larger RCF's can be generated by increasing the angularvelocity of the rotor assembly 200.

After the flow path 278 is established in the column 230 a, the fluidcan exit the column to establish a flow path 286 in the flow cell 232.The flow cell 232 includes optical windows, such as 282, and apertures284 that allow access to the optical windows. Next, the flow can exitthe flow cell 232 to establish a flow path 288 in the return segmentlink 220. As the flow progresses through the return segment link 220,the first flow path segment from the mixing chamber 228 to the returnsegment link 220 can fill with fluid and the flow can begin enteringinto the second flow path segment. The flow can then begin moving fromthe return segment link 220 towards a center of the rotor assembly 200and eventually reach the exit 250 of the adjustable tube 226.

In the second flow path segment where the flow is generally moving fromthe platter's 202 edge to the center axis 265, the fluid can exit thereturn segment link 220, to establish flow path 290 in a return flowchannel (More details of the flow path in the return segment link 220and a return flow channel are described with respect to FIGS. 5B and5C.). The flow can exit the return flow channel and begin filling thedrainage channel 248 to establish a flow path 292 in the drainagechannel. As described with respect to FIG. 4A, the flow 292 can receiveflow from a number of return flow channels associated with differentcolumns. Two gaskets can be used to prevent fluid in the drainagechannel 248 from escaping. Cross sections of gaskets, such as 275 a and275 b, are indicated in FIG. 5A.

In other embodiments, the second flow path segment can receive onlyfluid from the first flow path segment. For instance, the second flowpath segment can comprise flow conduits that receive only fluidassociated with the flow through the column 230 a and flow cell 232 andnot other columns. In this embodiment, additional return flow conduits(not shown) can be used. For instance, one manifold block 225 and oneadjustable tube 226 can be associated with one return flow channel fromeach column, such as 230 a, and the drainage channel 248 can beeliminated. As previously described, coalescing the flows from multiplecolumns in the drainage channel can reduce an amount of flow conduitsthat are used in the rotor assembly 200. One advantage of reducing theflow conduits can be reduced manufacturing costs.

After the flow enters the drainage channel 248, it can exit through oneor more apertures into one of the manifold blocks, such as 225, toestablish a flow path 294 within the manifold block 225. The manifoldblock 225 can be connected to adjustable tube 226. The flow can exit themanifold block 225 to establish a flow path 295 in the adjustable tube226. The flow path in the adjustable tube 226 can end at the exit 250.

Fluid can be introduced into the mixing chamber 228 until the fluid headthickness 268 reaches the same radial location 264 of the exit 250. Atthis point, if no additional fluid is introduced to the mixing chamber,to satisfy an incompressible Bernoulli's equation, the fluid headthickness 268 can reach equilibrium at about the same level of the exit250. Because of viscous and other non-linear effects, such as precessionof the rotor assembly about its center axis 265, the radial locations ofthe exit 250 and the fluid head thickness 268 may not be exactly thesame but can be at approximately the same distance. If additional fluidis introduced into the mixing chamber 228, the fluid head thickness canreach a radial location that is smaller the radial location of the exit250. When the fluid head thickness 268 is greater than its equilibriumvalue, the flow in the rotor assembly 200 can attempt to return to theequilibrium condition. As a result, flow can start moving out of themixing chamber 228 and flowing through the rotor assembly 200. Excessfluid can spill out of the exit 250.

If at some time, fluid introduction is stopped in the mixing chamber,then fluid can spill out of the exit 250 and into the drainage columnuntil the fluid head thickness 268 reaches about at the same radiallocation as the exit 250. When the fluid is continually introduced intothe mixing chamber drainage column 244, the flow can continually exitthrough each of the adjustable tubes and exit into the drainage column244 to establish a flow path 296 in the drainage column. The flow canexit the drainage column 244 through a bottom of the manifold assembly204 and eventually reach an interior of the reservoir, such as 210 a.Details of the flow path after it exits the manifold assembly 204 aredescribed in more detail with respect to FIG. 6.

The radial location of the fluid boundary surfaces associated with thefluid head and the exit 250 can vary. In the embodiment in FIG. 5A,during rotation, the centrifugal forces can cause the flow to build upagainst a gravitational pull such that it extends from a bottom of themixing chamber to top of the mixing chamber and from a bottom of theadjustable tube to the top of adjustable tube across each of the exits,such as exit 250. As a result of at least surface tension and gravityeffects, the boundary surface of the fluid head from the top to thebottom of the mixing chamber or the boundary surface of the fluid acrossthe exit 250 can be curved such that the radial distance from the centeraxis of rotation 265 can vary across the fluid boundary surfaces atthese locations. As the angular velocity of the rotor assembly isincreased, the radial variations along the fluid boundary surface candecrease, i.e., the surface can become more vertically aligned.

In one embodiment, to reach the equilibrium described above where thefluid head thickness 268 is at about the same radial location 264 as theexit (i.e., the fluid head thickness 268 is at about line 262), therotor assembly 200 can be spun up to a constant angular velocity (Also,as previously described, fluid can be introduced to the mixing chamberduring spin-up of the rotor assembly where the angular velocity of therotor assembly is increasing over time.). After the rotor assembly 200reaches some target angular velocity, fluid can be added to the mixingchamber 228 where it can progress through the rotor assembly 200 untilthe flow reaches the exit 250 and begins to flow out the adjustabletubes 226. A rate that fluid is added to the mixing chamber 228 can beused to control a flow velocity in the rotor assembly 200 including aflow velocity in the columns, such as 230 a and 230 b.

As previously described with respect to FIG. 1, after a determination ismade that a steady flow velocity is established in the columns, such as230 a and 230 b, a sample can be introduced at some location on therotor assembly 200. For example, the sample can be added to the mixingchamber 228. From the mixing chamber, the sample can be dispersed toeach of the columns. In another example, as is described in more detailwith respect to FIG. 9A, a sample can be injected near a head of eachcolumn, such as 230 a and 230 b. When the column includes achromatographic packing material, the sample can undergo achromatographic separation, which can be analyzed via measurement madeusing the flow cells at the end of each column, such as 232.

The equilibrium fluid head thickness 268 can be adjusted by changing thedistance 264 of the exit 250 from the center axis of rotation 265. Forexample, the equilibrium fluid head thickness can be selected so thatduring operation the fluid head does not extend into the aperture in theseat 240 of the mixing chamber 228. As previously described, theaperture in the seat 240 of the mixing chamber can provide an entrancefor one or more flow conduits that can deliver fluid into the mixingchamber 228.

In general, in various embodiments, a rotor assembly 200 can include 1)a first flow path segment where the flow moves along some flow path suchthat a final radial distance from the center 265 along the flow path isgreater than the initial radial distance and 2) a second flow pathsegment, connected to the first flow path segment, where a final radialdistance of the flow path segment is smaller than the initial radialdistance of the flow path segment. An exit can be placed at the finalradial distance of the second flow path segment such that fluid canleave the second flow path segment. The radial distance from the centeraxis 265 where the exit is located on the second flow path segment candetermine an equilibrium location in the first flow path segment. Fluidcan be introduced into the first flow path segment at some rate. Therate that the flow is introduced into the first flow path segment can beused to control a flow velocity through the first flow path segment andthe second flow path segment. As is described below, the fluidintroduction rate and associated flow velocity can be relativelyinsensitive to changes in the angular velocity of the rotor assembly200.

Returning to FIG. 5A, in yet other embodiments, the exit radius, such as264, of exit 250 of each of the adjustable tubes 226 can be adjusted.The exits, such as 250, for each of the adjustable tubes can be locatedat the same or different distances from the center axis 265 of the rotorassembly 200. The adjustable tubes, such as 226, can include threads 297that mate with threads in the manifold assembly 204 and threads (notshown) that mate with threads in the manifold block 225. The adjustabletubes can be screwed to different depths into the manifold block 225 tochange the radial distance 262 of the exit 250. A lock nut 224 can beused to fix be used to secure adjustable tube 226 such that the radialdistance of the exit 250 remains fixed during rotation of the rotorassembly 200.

In particular embodiments, the rotor assembly 200 can include amechanism that allows the radial distance of the exit 250 to bedynamically changed. For instance, the ends of each of the adjustabletubes, such as 226, can include a flexible end piece that is elasticallyextendable. In operation, a force can be applied to each of the flexibleend pieces to change the radial distance of the exit 250 associated withthe flexible end piece. For instance, when force is not applied to theflexible end piece, the exit 250 can be near the wall of the drainagecolumn 244 and when a force is applied, the exit 250 can be stretchedtoward the center of the drainage column 244.

An advantage of configuring the rotor assembly 200 with a first flowpath segment to the rotor edge connected to a second flow path segmentfrom the rotor edge is that once an equilibrium condition isestablished, the flow velocity can be controlled by changing the flowintroduction rate in the first flow path segment, such as but notlimited to via the mixing chamber 228. When ideal conditions areapproached, the flow introduction rate to the first flow path segmentneeded to obtain a particular flow velocity in the columns can beessentially independent of the angular velocity for a wide range ofangular velocity operating conditions.

Further, the flow introduction rate in the mixing chamber 228 needed toobtain a particular flow velocity in the rotor assembly 200 can be muchless sensitive to changes in angular velocity as compared to other flowconfigurations where the flow is not is routed back away from the edgeof the rotor. For example, the flow can be allowed to exit at the rotoredge, such as from return segment link 220, rather than allowed to exitat 250. In one embodiment, a fluid collection ring, such as 210, cansurround the rotor edge and receive fluid, such as fluid exiting fromthe return segment links (In this example, the return segment link doesnot turn the flow back towards the center 265 of the rotor assembly).For this configuration as compared to a configuration shown in FIG. 5Awhere the flow is routed back toward the axis of rotation 265, the exitvelocity at the rotor edge and the flow through the columns can be muchmore sensitive to changes in angular velocity. Typically, for this typeof configuration the exit flow velocity can increase as the angularvelocity of the rotor assembly is increased because RCF increases.

With respect to FIGS. 5B and 5C, further details of the flow near therotor edge are described. In particular, FIG. 5B is a top perspectivecross sectional view of the platter 202, column 230, which can containpacking material (referred to also as a stationary phase material) usedin a chromatographic process (not shown), a flow cell 232, a returnsegment link 220 and a return flow channel, such as 298 a and 298 b,with an illustrated flow path. The column 230 can rest on platter 202.The platter 202 can include a groove in which the column rests.

As previously described, a detachable cap 274 can be coupled to the endof the column 230. In one embodiment, the detachable cap 274 can includea protuberance that is configured to fit into an opening in the flowcell 232. A gasket 274 a can surround the protuberance to form a sealbetween the detachable cap 274 and the flow cell. The platter 202 caninclude a recessed portion into which the flow cell 232 can be placed.

The flow cell 232 can include an opening that allows fluid to travelinto the return segment link 220 (see 252 a and 252 b in FIG. 5C). Flowcan exit the channel in return segment link 220 and enter into a returnchannel, 298 a and 298 b. A gasket 220 a can be placed between thereturn segment link 220 and the edge of the platter 202. The gasket 220a can be configured to keep fluid from leaking at the interface betweenthe return segment link 220 and the flow cell 232 and the interfacebetween the return segment link 220 and the return flow channel, 298 aand 298 b.

The platter 202 can include an acceptor for a fastener 222 a. Thefastener can be inserted through an aperture in the return segment link220. Tightening the fastener can increase a force on the gasket 220 abetween the return segment link 220 and the platter 202. The force onthe gasket 220 a can provide better seal integrity, which can preventleakage. The return flow channel can include a portion 298 a that goesinto the platter 202 through an outer edge of the platter 202 and aportion 298 b that goes down through the platter (portion 298 a and 298b are at angle to one another). The portion 298 b can exit into thedrainage channel 248.

FIG. 5C is a perspective view of an embodiment of a return segment link220. The return segment link 220 can include apertures 300 and 306 thatallow the return segment link 220 to be coupled to two other returnsegment links. A pin can be inserted through each of the apertures 300and 306 to couple the return segment link to the other return segmentlinks. When all of the return segments links are coupled together, anunbroken chain is formed around the edge of the rotor.

The return segment link 220 can include an aperture 304. As illustratedin FIG. 5B, a fastener can be inserted through the aperture 304 tocouple the return segment link to the platter 202. In one embodiment,the return segment link 220 can include two recessed portions 302 a and302 b. Each of the recessed portions can allow flow between a flow celland a return flow channel in the platter 202. Thus, in one embodiment,each return segment link 220 can be associated with the flow from twodifferent chromatographic columns.

An inner surface of the return segment link 220 can be curved. Thesurface curvature can be selected such that the when the return segmentsare linked, the inner surfaces of the return segment links 220 form acircle that is slightly larger than the circle around the edge of theplatter 202. With the surface curvature 308, the forces generated fromthe fastener that is attached through aperture 304 can be betterdistributed. A better distribution of forces over surface 308 canimprove the seal integrity between the return segment link 220 and theouter edge of the platter 202.

FIG. 6 is a side view of the rotor assembly 200 with a cross-sectionalview of a reservoir 210 and a partially transparent view of the adapterplate assembly 214. The adapter plate assembly 214 can include anadapter plate 318. The manifold assembly 204 can be coupled to theadapter plate 318 via fasteners, such as 320. The tapered shaft 212,which can be connected to a motor (not shown), can be coupled to a diskportion 214 a of the adapter plate assembly 214 via a fastener 316. Thetapered shaft 212 can be inserted into a bottom portion of the adapterplate assembly 214 and secured to it via the fastener.

The reservoir 210 can also be coupled to the adapter plate assembly 214,such as to the disk portion 214 a of the adapter plate assembly. Theadapter plate 318 can include one or more flow conduits. The flowconduits can allow flow exiting from the drainage column 244 in themanifold assembly 204 to enter into the adapter plate. One or moregaskets (not shown) can be used for sealing purposes where the fluidinterface on the manifold assembly 204 meets the fluid interface on atop of the adapter plate 318.

Fluid can enter from the manifold assembly 204 into the adapter plate318 and then travel through one or more flow conduits to exit theadapter plate via one or more apertures 310. In a particular embodiment,the flow can travel in an increasing radial direction from the center ofthe rotor assembly to enter into the reservoir 210. The flow can thentravel into an interior portion of the reservoir, such as 210 a.

A seal can be formed at the fluid interface between the reservoir 210and the adapter plate 318. In one embodiment, a channel can run aroundan edge of the adapter plate 318 which can be sealed by the gaskets 314a and 314 b. An advantage of this approach is that the adapter plate 318can be inserted into an interior portion of a single piece reservoirdrum, such as 210. The reservoir drum 210 can include a circular ledge322 or a number of arms that extends from an inner surface of thereservoir drum. The adapter plate 318 can rest on this ledge or thearms. The disk portion 214 a of the adapter plate assembly can beconfigured to be inserted through the circular ledge 322 or arms. Thecircular ledge or arms can include mounting points for attaching thereservoir drum to the adapter plate 318.

In particular embodiments, the reservoir drum can include multiplepieces, such as two halves or four quarters. The adapter plate 318 caninclude flow conduits that extend from the adapter plate 318 that areconfigured to be inserted into apertures in the reservoir drum 210 orthe reservoir drum 210 can include flow conduits that extend from thereservoir drum and can be inserted into the adapter plate. The reservoirdrum 210 can be formed in multiple pieces to allow for assembly. Thisconfiguration can be used an alternate to using the sealed channelaround the edge of the adapter plate.

Different rotor assemblies can have different manifold assembly designsand different waste reservoir designs. For example an inner diameter ofthe waste reservoir drum 210 or an outer diameter of the manifoldassembly 204 can be larger. Different adapter plates 318 can be used toaccommodate the different configurations of the manifold assembly 204 orthe reservoir drum while allowing a portion of the adapter plateassembly that is coupled to the tapered shaft 212 to be reused.

The rotor assembly described with respect to FIGS. 5A, 5B, 5C and 6comprises a number of modular components. The modular architecture isprovided for the purposes of illustration only and is not meant to belimiting. An advantage of some modular architectures can be ease ofassembly, disassembly, more configuration options and reducedmanufacturing costs. A disadvantage of some modular architectures can bethat more modular components can introduce more fluid interfaces thatnecessarily need to be sealed and thus, more potential locations forleaks.

To eliminate potential leak points, in some embodiments, some componentscan be integrally formed to perform functions associated with twoseparate components. For instance, in some embodiments, a manifoldassembly can include a waste reservoir that is integrally formed withthe manifold assembly to eliminate the flow conduits through the adapterplate 318. As another example, the mixing chamber enclosure is formed asa separate component from the platter 202. In other embodiments, themixing chamber enclosure can be an integral component of the platter202. Further, the manifold block 225 and adjustable tube 226 can beformed as a single flexible conduit.

In yet another example, the platter 202 includes a number of grooves onwhich columns can be rested. Thus, the columns can be removed, such asfor the purposes of packing or cleaning the columns and then be placedback on the platter. In other embodiments, the columns can be integrallyformed into the platter 202. For instance, a solid disk from which theplatter is formed can have a number of holes bored into it that canserve as columns. In another embodiment, metal can be poured over aconduit structure that can be subsequently removed to provide aninternal conduit system within a platter such as 202.

In yet another embodiment, a number of open channels can be formed on afirst plate and then the channels can be covered. For instance, a secondplate can be adhered to the second plate to cover the channels. Afterthe channels are covered each channel has an entrance and an exit. Forinstance, a number of radially aligned channels directed can be formedon a first “washer” shaped disk where each channel progresses from aninner radius to an outer radius of the washer. Then, a second structure,such as a second washer of a similar dimension of the first washer witha flat surface can be adhered to the first washer to form enclosedchannels. In particular embodiments, open channels can be formed in eachof two separate plates, such as forming half cylinder channels in eachof the plates. When the two separate plates are joined, enclosedchannels with a circular cross section are formed.

FIGS. 7A-7C are side and perspective views of a gas bearing supportassembly 400. The gas bearing support assembly 400 can be placed upon adrum type rotational mechanism that rotates a rotor assembly (see FIG.8). The lid 414 can act as a lid to the drum. The lid can be composed ofa hard transparent material, such as bullet-proof glass. The bulletproof glass can allow the operation of the rotor assembly to be observedwhile providing protection from parts that can be flung from the rotorassembly as a result of a mechanical failure of some type. In otherembodiments, the lid can be formed from another type of material, suchas non-transparent material.

During rotation of the rotor assembly 200, the gas bearing 418 can reston the seat 240 of the rotor assembly, such as 200. The weight of thegas bearing support assembly 400 that is transferred through the gasbearing 418 and onto seat 240 can help stabilize a rotor assembly, suchas 200, during rotation. For example, the weight of the gas bearingsupport assembly 400 can help to lessen precession effects that canoccur while the rotor assembly is rotating.

The gas bearing support assembly 400 can include a number of flowconduits. The flow conduits 416 can run through a hollow shaft 412 andextend through an aperture in a center portion 426 of the gas bearing418. As previously described, when the gas bearing 418 rests on seat240, the flow conduits can extend into a chamber located below the seat240, such as a mixing chamber 228. Further, the bottom of the gasbearing 418 can act as a portion of a lid to the mixing chamber to helpcontain fluid in the mixing chamber.

In a particular embodiment, three flow conduits 416 are shown extendingthrough the gas bearing assembly. The three flow conduits can be used todeliver a variety of fluids to the mixing chamber, such as two differentsolvents and a sample fluid. The concentrations of the differentsolvents can be varied over time to form an elution gradient. In otherembodiments, more or less flow conduits can be run through shaft 412 andinto the mixing chamber. In some instances, a gas bearing assembly canbe used for the purposes of rotor assembly stabilization and may notinclude any flow conduits. In another instance, for a particular run,one or more flow conduits can be inactive and not utilized.

The shaft 412 can include one or more fluid interfaces allow one or moreflow conduits from the solvent reservoirs 285 and/or the sampleintroduction mechanism 402 to be connected to the flow conduits runningthrough a center portion of the shaft. For instance, the shaft 412 canbe coupled to a cap 406 with interfaces that can connect to flowconduits, such as flexible tubing, from the solvent reservoirs 285 andthe sample introduction mechanism 402. One or more pumps associated withthe solvent reservoirs and the sample introduction mechanism 402 canmove fluid into the shaft.

In one embodiment, one or more of the pumps can be a positivedisplacement pump, such as a piston driven pump. In another embodiment,one or more of the pumps can be positive displacement pump, such as abuoyancy pump. A compressed-air-powered double-diaphragm pump is oneexample of a buoyancy pump. This type of pump can run on compressed airand has a minimal amount of moving parts. In yet another embodiment, thepump can be a gas amplifier pump.

The gas bearing 418 can be porous. The pores in the gas bearing allowgas to flow through it. When the gas bearing 418 rests on seat 240 andthe rotor assembly is rotating, the gas pumped through the gas bearing418 can form a thin layer between the gas bearing 418 and the rotatingseat 240. The thin layer of gas minimizes friction between the gasbearing 418 and the seat 240. Some times the seat 240 and the gasbearing can come into contact. This contact can eventually wear out thegas bearing 418.

The gas bearing can include an interface to a gas source 404, such as apressurized argon container. During operation, the pressurized argon canflow through the gas bearing 418. An advantage of argon is that it isthe third most common gas in the atmosphere and it is inert. However,other gases can be used and Argon is provided for the purposes ofillustration only.

The gas bearing support assembly 400 can be configured to allow theshaft 412 and the gas bearing 418 to be raised or lowered, 422. The gasbearing support assembly is shown in a raised position in FIG. 7A and alowered position in FIG. 7B. In one embodiment, the gas bearing supportassembly can be raised or lowered via lever 408. The lever 408 cancoupled to a rotatable shaft 410 where a rotation 420 of the shaft 410raises or lowers the shaft 412 and the gas bearing 418. The rotatableshaft 410 can be supported by support block 424. The support block 424can be coupled to the lid 414 via one or more fasteners, such as 428.

FIG. 8 is side view of a rotor assembly 200, gas bearing supportassembly 400 within a containment structure 440 and including instrumentmounts, such as 444 and 442. The gas bearing support assembly 400 isshown in engaged position where the gas bearing 418 rests on the seat240 of the rotor assembly 200. The gas bearing lid can rest on a ledgeof the containment structure 440.

In one embodiment, the containment structure 440 can be drum shaped andcan be formed from a metal. The containment structure 440 can include anaperture that allows the tapered shaft 212 to be coupled to a motor thatis located below the rotor assembly 200. The motor can be used to impartan angular velocity to the rotor assembly 200.

One or more apertures can be provided in the containment structure orthe lid 414 (not shown). The apertures include conduits that allowgasses to be evacuated from the chamber 450. In one embodiment, thechamber 450 can be sealed so that a vacuum of some level can beestablished in the chamber. To improve sealing, a gasket can be placedbetween the lid 414 and the containment structure 440. Further,fasteners can be used to more securely couple the lid 414 to thecontainment structure. The one or more apertures in the lid 414 or thecontainment structure 440 can be connected to a vacuum pump. When thevacuum pump is activated a vacuum of some level can be established inthe chamber 450. The vacuum pump can also be used for safety purposes tolimit an accidental build-up of dangerous gasses in the chamber 450,such as flammable gas.

One or more instrument mounts, such as 442 and 442, can be coupled tothe containment structure. In one embodiment, each instrument mount,such as 442 and 444, can include a light source and a light gatheringdevice. The light source and light gathering device can be aligned withthe flow cell windows on the rotor assembly 200 as previously described.To allow the light source and the light gathering device to be alignedwith the flow cells, which are near the edge of the rotor assembly 200,the instrument mounts can extend over and under a portion of the rotorassembly 200.

In a particular embodiment, two fiber optic cables can be attached toeach instrument mount, such as 442 and 444. A first fiber optic cablecan be coupled to a light source, such as light source that emitsphotons at one or more different wavelengths. A second fiber optic cablecan be coupled to a photomultiplier tube. The second fiber optic cablecan receive photons emitted from the first fiber optic cable that havepassed through a flow cell. The gathered photons can be passed to aphotomultiplier tube. The photomultiplier tubes and the lights sourcescan be located outside of the containment structure 440.

The signal generated from the photomultiplier tube can be used toanalyze constituents of the fluid passing through the flow cell. Thelight sources associated with each instrument mount can producedifferent wavelengths, such as but not limited to a visible wavelengths,ultra violet wavelengths and infrared wavelengths. In variousembodiments, zero, one or more than two instrument mounts with lightemitting and light gathering capabilities can be used.

In another embodiment, the instrument mounts, such as 442 and 444, caninclude gas bearings. The gas bearings can help to stabilize the rotorassembly 200 during rotation and can help to prevent the rotor assemblyfrom colliding with the instrument mounts, such as 442 and 444. A flowconduit, such as flexible tube, can be coupled to each of the instrumentmounts to provide gas for the gas bearings. The flow conduit can becoupled to a gas source that is used with each of the gas bearings.

The instrument mounts, such as 442 and 444, can include a rotatablejoint that allows a portion of the instrument mount to rotate. Via therotatable joint, a portion of the instrument mount, such as the upperportion of the instrument mount can be rotated, such as rotated upwards,to allow the rotor assembly to be placed in or removed from thecontainment structure 440.

In one embodiment, when there is enough clearance between the sides ofthe containment structure and the edge of the rotor assembly, a portionof the instrument mount, such as the portion including the fiber opticcables can be located on retractable arms. The retractable arms can beretracted to allow a rotor assembly to be placed in or removed from thecontainment structure. The retractable arms can be extended to allow thefiber optic cables or any other instrumentation on an instrument mountcoupled to the retractable arms to be placed in a position above and/orbelow the rotor assembly 200.

As the rotor assembly rotates, various rotor locations can rotate in arepeating manner past the instrument mounts. To keep track of thecurrent rotor location, such as a rotor location at which a measurementis made. An indexing system can be provided. The indexing system can beused to uniquely identify a location where a measurement is made on therotor assembly. A generated index can be associated with a measurementmade using the instrumentation associated with the instrument mounts.

As an example, the rotor assembly 200 can include an identifier such asmarkings that allow a rotor location to be determined. For instance, theplatter 202 of the rotor assembly 200 can include visible markings thatuniquely identify a location, such as location where each column isplaced. The marking can be a symbol, such as a number and/or letter or abar-code. As another example, RFID tags storing an identifier can beplaced at various locations around the edge of the rotor assembly 200,such as a location where each column is placed.

A detector can be located on the instrument mount that is configured todetect the identifiers as they pass by the instrument mount. Forinstance, a camera can be used to detect an identifier, such as a symbolor a bar-code. As another example, a laser and a detector can be used toread an identifier, such as a bar-code. In yet another example, an RFIDtag reader can be used to receive an identifier from an RFID tag placedon the platter.

During operation, many measurements can be made. The measurements can beassociated with a particular chromatographic column. The amount ofmeasurements that are made can depend on an angular velocity of therotor assembly and length of time over which measurements are made. Adetected identifier can be used as an index to map a set of measurementsto a particular feature of the rotor assembly, such as to a particularcolumn and associated flow cell. The indexed measurements can be used togenerate a time varying profile of the measurements associated aparticular chromatographic column.

FIG. 9A is a top view of an embodiment of a rotor assembly configuredfor use in a chromatographic system. Parts of the rotor assembly havebeen previously described with respect to FIG. 3. A power generationring 333 can be coupled to the rotor assembly. The power generation ring333 can include components of an electric generator, such as one or morecoils of wire, and voltage conversion circuitry.

When used as an electric generation, the power generation ring 333 canbe moved past magnets, such as magnets mounted on the instrument mountsdescribed with respect to FIG. 8. As the coils move past the magnets, acurrent can be induced in the one or more wire coils. The generatedelectricity can be used to power devices on the rotor assembly. Inanother embodiment, the power generation ring 333 can include a numberof batteries that are evenly distributed around the rotor assembly forbalance. These batteries may or may not be chargeable via an electricgenerator located on the rotor assembly.

In yet another embodiment, a brush interface can be used to transferpower to the rotor assembly 200. For example, a conductive stripped canbe placed around an edge of the containment structure 440 shown in FIG.8. One or more arms with conductive brushes or some other contactmechanism can be extended from the rotor assembly 200 such that theconductive brushes contact the conductive strip, which is stationary, todeliver power to the rotor assembly. The conductive strip can beinsulated so that the entire device is not electrified. In anotherexample, a portion of the rotor assembly 200, such as the tapered shaft212 can include an insulated conductive strip. Conductive brushes, whichare stationary, can be located within the containment structure 440 thatcontact the insulated conductive strip on the rotor assembly.Electricity can be transferred between the metal brushes to the rotorassembly while it is rotating to power various devices located on therotor assembly.

The voltage conversion circuitry can condition electricity received froman off-rotor source or power generated on-rotor for use with variousdevices located on the rotor assembly 200. One or more electricalconduits can lead from the power generation ring 333, such as conduit334 to allow various devices to receive power. The conditionedelectricity can be used to power electronically actuated valves,communication devices, on-rotor instrumentation, on-rotor sensors andenvironmental control devices, such as heating elements as well as tocharge on-rotor batteries.

In FIG. 9A, the electrical conduit 334 is shown coupled to heatingelements, 329 and 331, and to a sample introduction mechanism 325. Theheating element rings, such as 329 and 331, can include a singleelectrical heating coil or multiple electrical heating elements thatreceive power from the power generation ring 333. A controller can beassociated with one or both of the heating element rings, i.e., onecontroller for both rings or a separate controller for each ring. Theone or more controllers can be configured for controlling a heat outputof each of the heating element rings. In one embodiment one or both ofthe heating element rings can include one or more heating elementsassociated with each column where the heating elements for each columncan be individually controlled. Temperature sensors can be located alongthe columns and control, such as a target temperature value, can bebased upon data received from the temperature sensors.

In one embodiment, a sample introduction mechanism 325 can be located ona rotor. For instance, the sample introduction mechanism 325 can includea fluid interface, such as an electronically actuated valve, thatconnects a sample reservoir in the sample introduction mechanism to eachof the columns and allows samples stored in the sample reservoir to beinjected into each column. The sample introduction mechanism 325 caninclude one or more on-board injectors, such as one or more pumps, thatallow a fluid stored in the sample reservoir to be injected into eachcolumn. In one embodiment, the injector can be a syringe pump.

In addition, the sample introduction mechanism can include one or morerefill ports, such as 327, that are each connected to a samplereservoir. The refill ports can allow the sample reservoirs to berefilled. In particular embodiments, a single sample reservoir canprovide samples to multiple columns. For instance, one sample reservoircan supply two or more columns where a single refill port, such 327, isassociated with the reservoir. Further, the sample used in each samplereservoir can vary from reservoir to reservoir. Thus, during operation,a number of different samples can be chromatographically processedsimultaneously on the rotor. In other embodiments, when multiple samplereservoirs are used, the sample reservoirs can include separatelycontrolled injectors. The injectors can be separately controlled toallow different samples to be introduced at different times relative toone another. The sample introduction mechanism 325 can include acontroller for controlling devices located on the sample introductionmechanism, such as sample injectors or controllable valves.

The rotor can include on-board circuitry 337, such as processor, memory,battery and/or a communication interface. In one embodiment, circuitry337 can include a wireless communication interface that allowscommunication between the rotor and a remote device, such as a systemmanagement device, such as the system management described with respectto FIG. 1. The circuitry 337 can include a communications bus thatallows data to be transmitted from the circuitry 337 to the variousdevices. For instance, the circuitry 337 can receive commands from aremote device that are transmitted to a particular device on the rotorassembly, such as the power generation ring 333, heating element rings,329 or 331, the sample introduction mechanism 325 or an adjustablevalve.

A number of devices that generate data on-board the rotor can beconnected to the communications bus 341 via a wired communication link,such as 339. In other embodiments, wireless communication links can beused to provide communications between the various devices on the rotor.An advantage of wireless links is that the wiring paths through therotor assembly can be minimized. For example, various devices caninclude wireless transmitters/receivers that allow for communicationswith the circuitry 337.

Devices can generate data that is sent to the circuitry 337 via wirelessand/or wired communication links. The circuitry 337 can store and/orsend the data to a remote device. For example, the rotor can include anumber of sensors such as but not limited to temperature sensors, flowrate sensors, flow level sensors (e.g., the reservoir 210 or the mixingchamber 228 can include a flow level sensor), pressure sensors andcombinations thereof. Data from the sensors can be transmitted to aremote device via the circuitry 337. In other embodiments, particulardevices can include their own communication interface that is notconnected the circuitry 337. For instance, the sample introductionmechanism 325 can be configured to communicate directly with a remotedevice rather than through an intermediary device, such as circuitry337.

As another example, the rotor can include on-board instrumentation thatgenerates measurement data. The on-board instrumentation can communicatewith circuitry 337 to send data to a remote device. In some embodiments,the data can be sent in real-time, i.e., as it is generated. In otherembodiments, one or more devices can include a memory that allows datato be stored and then later transmitted to a remote device. The memorycan also serve as a back-up data source where data is both transmittedand real-time and stored to memory, such that if any of the real-timedata is lost, such as due to a transceiver malfunction, data can berecovered from the memory.

FIG. 9B is a side view of an embodiment of a rotor assembly configuredfor use in a chromatographic system including a fraction collectionmechanism 350. In general, the fraction collection mechanism receiveseluents eluted from a chromatographic enclosure. Thus, the fractioncollection mechanism can be considered an eluent reservoir. The locationof this fraction collection mechanism is provided for illustrativepurposes only. Different rotor configuration can have different columnand flow path configurations where a different placement of the fractioncollection mechanism may be utilized.

In one embodiment, a fluid interface, such as a valve 352, can connectthe fraction collection mechanism to flow path located in a return flowchannel on the platter 202. As is described with respect to FIGS. 5A and5B, the return flow channel can be coupled to an exit of a flow cell,such as 232 via a return segment link 220. The valve 352 can be openedand closed at various times to allow fluid that has exited the flow cell232 to enter into the fraction collection mechanism 350 or by-pass thefraction collection mechanism 350. The valve 352 can be opened andclosed while the rotor assembly is rotating.

In particular embodiments, the valve 352 can receive commands to openand close that are initiated on a device that is located off of therotor. The remote device based upon information, such as measurementsmade via the flow cell, can determine when to open and close the valveand then send a command that causes the valve to open or close. Inanother embodiment, a controller located on the rotor assembly can makethis determination and in response can send a command to the valve 352to either open or close.

The fraction collection mechanism 350 can include one or more chambersfor storing collected fractions. For instance, the fraction collectionmechanism 350 can include four chambers 356 with four valves 354 thatare individually controllable to direct fluid to each of the fourdifferent chambers. The chambers, such as 356, can each include anaccess aperture that allows a collected fraction to be extracted. Forinstance, in some embodiments, fractions can be collected via theapertures, after a run is completed and the rotor assembly is at rest.

A fraction collection mechanism, such as 350, can be associated with oneor more columns. For instance, a single fraction collection mechanismcan be configured to receive fractions from a single chromatographiccolumn or can be configured to receive fractions from multiple columns.The number of fraction mechanisms on the rotor assembly can varydepending on a mapping between each fraction collection mechanism and anumber of columns it serves as well as a total number of columns on therotor. Further, when fraction mechanisms are employed, a fractioncollection mechanism does not have to be associated with every column onthe rotor. Thus, a number of fraction collection mechanisms on the rotorassembly can vary for different rotor assembly configurations.

In yet other embodiments, a variable volume eluent capture device, suchas 350, can come after the detectors, such as a flow cell. A variablevolume eluent capture device can include a wireless remote controlledelectric motor that turns a lead screw inside a chamber, such as acylindrical chamber. Onto the lead screw can be attached a piston-likesealing washer. As the lead screw turns, the piston-like sealing washercan move towards the outside of the chamber thereby allowing liquid toflow at a rate that is affected by the rate of movement of thepiston-like sealing washer.

A variable volume eluent capture device can be incorporated into thereservoir, such as 210, previously described. In another embodiment, adevice, such as a reverse syringe pump, can be used as a variable volumeeluent capture device. In some cases, it can desirable to have multiplechamber variable volume eluent capture devices on the end of each columnsuch that fraction collection is possible. In this case, a multiportvalve can be added between the column and the variable volume eluentcapture devices.

FIG. 9C is a block diagram including a reaction chamber located betweenthe end of a column, such as 232 and a detector or structure associatedwith a detector, such as flow cell. In some instances, after achromatographic separation, a chemical reaction can be performed onanalyte leaving a chromatographic enclosure, such as column 232. Thereaction can be performed to allow the analyte allow to be betteridentified with a particular detector. For instance, if an analyte,pre-reaction, does not fluoresce, it can not be detected by afluorescence detector, such as a LIF detector (see Instrumentationsection below). Via a chemical reaction, the analyte can be altered toproduce products with enhanced detection characteristics, such asproducts that are detectable with a particular type of detector.

In one embodiment, a flow diverter, such as 800, can be used to divert aflow leaving the column, such that it either moves directly to adetector, such as flow cell 232 or is diverted into a reaction chamber,such as 802, before it reaches the flow cell. The reaction chamber 802can include access ports that allow one or more different reagents, suchas 804 and 806, to be added alone or in combination to the reactionchamber 802. The access ports can be controlled by valves, such as 808and 810. In embodiments described herein, all or a portion of thecolumns can include reaction chambers, such as 802. Further, a reactionchamber, such as 802 can receive an analyte from one or more differentcolumns.

The reaction chamber can include an ultrasonic vibration generator. Inone embodiment, the ultrasonic vibrations can be generated by apiezoelectric element. The ultrasonic vibrations can enhance reactionrates of the analyte and reagent ensuring completeness of the chemicalreaction. In one embodiment, the reaction chamber 802 can be locatedbetween the column 230 and flow cell without use of a flow diverter,such as 800. In this embodiment, reagents can be added to the reactionchamber when desired or the flow can be allowed to pass without addingreagents. In yet other embodiments, multiple reactions chambers can beused, such as a series of reaction chambers where a multi-step reactionis performed.

In other embodiments, another type of energy source can be used toaffect a reaction, such as to increase a reaction rate in the reactionchamber 802. For instance, a microwave source can provide microwaves tothe reaction chamber. In another example, a temperature controlmechanism can be used to heat or cool the reaction chamber as needed.

With respect to FIGS. 10A-D, an alternate system for performingcentrifugal liquid chromatography is described that incorporates aswinging bucket design. FIG. 10A is a front and side view of a bucketassembly 500 including a plurality of columns for performingchromatographic separation. The bucket assembly is detachable. Inoperation, as is shown in FIGS. 10B-D, the bucket assembly 500 can becoupled to a rotor assembly and rotated. The rotation can impartcentrifugal forces to the bucket assembly. The bucket assembly can becoupled to the rotor assembly via a rotor support 506. During rotation,the centrifugal forces can drive fluid down a number of chromatographiccolumns in which a chromatographic separation is performed.

The bucket assembly 500 can include a solvent reservoir 502 that can bepre-loaded with a solvent and a sample mixture. The solvent reservoir502 can be coupled to a large number of chromatographic columns 508 viaa well plate 504. During rotation, the solvent and sample can be driventhrough orifices in the well plate 504 via centrifugation. In oneembodiment, the well place can include 96 orifices coupled to 96columns.

In another embodiment, each bucket assembly can be coupled to a centralreservoir associated with the rotor assembly that rotates with thebuckets. The central reservoir can be located near an axis of rotationof the rotor assembly (see FIG. 10C). The central reservoir can controla fluid mixture delivered to each rotor assembly including a solventcomposition for an elution gradient and a sample. In a particularembodiment, a flexible fluid interface, such as a flexible hose, can beattached from central reservoir to the solvent reservoir 502 on eachbucket, such that fluid from the central reservoir can be delivered tothe solvent reservoir 502. As will be described below, a position of thebucket assembly can change during spin-up from a vertical position to ahorizontal position. The flexible fluid interface can be used toaccommodate a change in position of the bucket assembly during spin up.

A pack 510 including a battery and a transceiver can be coupled to thebucket assembly 500. The battery can be used to power a flow ratecontrol device 514. In one embodiment, the flow rate control device 514can be a motorized stopcock. The motorized stopcock can be coupled toeach of the columns. When the stopcock is closed liquid does not flowthrough the columns 508. When the stopcock is partially open, liquid canflow through the columns 508. When liquid starts moving down the column,air displaced from the columns can be released via vents, such as 516.

A position of the stopcock can be used to affect a flow rate in each ofthe columns. As the stopcock is more fully opened, the flow rate can beincreased through the columns. In one embodiment, a flow rate controldevice 514, such as the stopcock can be remotely controlled. A remotedevice can send commands to request a particular stopcock position viathe transceiver. Data associated with flow rate control device 514, suchas its current state, can be sent to a remote device via thetransceiver.

In a particular embodiment, a sample reservoir can be located above thesolvent reservoir 502, separate from the solvent reservoir, so that thesample can initially be kept separate from the solvent. A mechanism,such as a valve can be used that allows the sample to be introduced intothe solvent reservoir. In operation, the solvent can first be allowed topropagate through the columns to establish a flow in the columns. Then,the sample can be introduced into the solvent reservoir, such as 502,and a mixture of solvent and sample can be allowed to propagate down thecolumns 508. The solvent reservoir 502 can include a mixing mechanism,such as an ultrasonic mixer, that mixes the solvent and sample, afterthe sample is introduced.

A number of flow cells 512 can be located near the end of the columns508. The flow cells can be used to perform a number of opticalmeasurements of flow exiting the columns, 508. Details of some of theoptical measurements that can be made are described as follows withrespect to FIGS. 10B-10D. After exiting the flow cells, waste can becollected in the solvent receptacle 518.

FIG. 10B is a top view of a rotor assembly 525 including a plurality ofbuckets, such as 500. The top view is during rotation of the rotorassembly 525 and the buckets. The rotor assembly 525 can be configuredto rotate around an axis 501. The rotor assembly 525 can include anumber of mounts, such as 520, for attaching the bucket, such as 500. Inthe embodiment of FIG. 10B, the rotor assembly 525 can include 4 mountsfor attaching 4 separate buckets, such as 500, if desired. The rotorassembly 525 can be surrounded by a containment structure 534, such as ametal drum.

At rest, the buckets, such as 500, can be aligned vertically with thegravity vector (see also FIG. 10B). During operation of the rotorassembly 525, as its rotational speed increases, the bucket 500 canswing up to a horizontal position as shown in FIG. 10B. In a horizontalposition, the transceivers on the bucket can pass over a transceiverlocation 524 located on a bottom portion of the containment structure534. Via the transceiver location 524 data transmitted from each of thebuckets can be sent to a remote device or a remote device can send dataor commands to each of the buckets, such as commands to a flow controldevice located on each of the buckets.

In alternate embodiment, a number of buckets, such as 500, can bemounted to a platter, such as platter as described with respect to FIGS.2-8 so that the bucket is always in a horizontal position. Utilizing aplatter design, the buckets can be connected to a fluid reservoir, suchas a mixing chamber 228, for solvent and sample introduction and can beconnected to another flow path that allows waste to be collected fromthe buckets in a reservoir, such as reservoir 210 previously described.Further, the bucket can be configured in flow path where a velocitygenerated by the flow introduction rate is substantially independent ofthe angular velocity, i.e., where the flow moves from the center to anedge of the platter and then returns back towards the center.

Two instrument assemblies are shown. Each instrument assembly caninclude a mount, such as 530, which is attached to the containmentstructure 534, a retractable arm 528 coupled to the mount and one ormore detector elements, such as 526, coupled to the retractable arm 528.The arm 528 is in an extended position while an arm 532 is in aretracted position. When the arm 528 extended position, the detectorelements 526 can located over an optical flow cell line 522 so thatmeasurements involving the flow cells can be made. When an arm, such as532, is in a retracted position, then instrumentation associated withthe instrument assembly may not be used.

In some embodiments, a plurality of instrument assemblies can beavailable. However, every instrument assembly and its associatedinstrumentation may not be used for every run of the chromatographicsystem. When an instrument assembly is not in use, it can stowed, suchas in a retractable position, as shown in FIG. 10B.

FIG. 10C is a top view of a rotor assembly including a plurality ofbuckets at rest. FIG. 10D is a top view of a rotor assembly including aplurality of buckets during rotation. The rotor assembly includes acentral reservoir 570. The central reservoir can control fluidcomposition for each of the buckets. Fluid from the central reservoir570 can be delivered to the buckets via a flexible fluid interface 572.The flexible fluid interface 572 can include a detachable connector 574that can be linked to an acceptor on the bucket.

In particular embodiments, the central reservoir 570 can includemultiple chambers for storing different mobile phase fluids. Further,the reservoir can include a gradient former and a mixing chamber. Thegradient former can control a composition of a mobile phase fluid usingdifferent fluids. In addition, the central reservoir can include asample introduction mechanism and one or more sample reservoirs arrangedto introduce a sample. The sample can be combined with a mobile phasefluid in the mixing chamber. The mixing chamber can be in fluidcommunication with each of buckets via the flexible fluid interfaces,such as 572.

In one embodiment, a flexible power interface (not shown), such as powercable, can be coupled to the flexible fluid interface 572. The flexiblepower interface can be attached to an acceptor on the bucket to deliverpower to devices, such as controlled valves on the bucket. The flexiblepower interface can be coupled to a central portion of the rotorassembly. As previously described with respect to FIG. 9B, duringrotation, a brush interface can be used to deliver power to a rotatableelement from a power source that is located off of the rotatableelement.

A brush interface can be used to deliver power to a central portion ofthe rotor assembly while it is rotating. Thus, the buckets can alsoreceive power from the off-rotor power source via the flexible powerinterface between the central portion of the rotor assembly and each ofthe buckets. The off-rotor power source can also be used to powerdevices associated with the central reservoir 572. In anotherembodiment, an on-rotor power source can be used, such as an electronicgenerator or batteries, to power the central reservoir 572 and deviceson the buckets.

The configuration in FIGS. 10C and 10D includes 7 instrument mounts fordifferent types of detectors, 550, 552, 554, 556, 558, 560 and 562.During operation all or portion of the detectors can be utilized. InFIG. 10D, for one embodiment, all of the detectors can be utilized.Thus, all of the arms associated with the detectors are shown as beingengaged in FIG. 10D. In this instance, for each revolution of the rotorassembly, seven different measurements can be made for each flow celland then repeated for each subsequent revolution.

In particular embodiments, detector 550 can be a visible and UVabsorbance detector. Detector 552 can be a fluorescence detector.Detectors 554, 556, 558, 560 and 562 can be Laser-induced fluorescence(LIF) detectors. For each of the LIF detectors, a laser with a differentwavelength can be employed. Further details of instrumentation, such asdetails regarding an absorbance detector, a fluorescence detector and aLIF detector are described in the following section.

Instrumentation

After a sample has undergone a chromatographic separation, such as oneperformed during rotation of one of the column as previously described,one or more detectors can be employed to identify eluents that haveexited the column. In chromatography, matching a retention time of aneluent in the column to the retention time of a known eluent does notnecessarily provide sufficient information to identify the eluent. Thus,additional detectors can be employed.

In chromatography, the combination of a given substance's retention timefor a specific set of chromatographic parameters and the responsecharacteristics of that specific substance for a given detector can formthe basis of determining the chemical identity of that substance. Sinceall substances respond differently for each type of detector, the moredetectors used, the more certainty there can be about the chemicalidentity of the substance. When these two parameters are combined withthe retention time relative to one or more reference substances, thechemical identity can be determined with near certainty

The detector can be a system component that emits a response due to thedetection of an eluting sample compound and subsequently signals a peakon the chromatogram. Typically, the detector can be positionedimmediately posterior to the stationary phase in order to detect thecompounds as they elute from the column. For example, as describedabove, flow cells can be used with many types of optical detectors.Thus, the flow cell can be typically positioned at the end of thecolumn.

Information generated from a detector can often be displayed as a timechanging value, such as a curve with peaks and valleys that change overtime as different sample components elute from a column. The width andheight of the peaks can usually be adjusted using the coarse and finetuning controls associated with the detector. Also, the detection andsensitivity parameters associated with a detector can often becontrolled.

As previously described, in some embodiments, a detector and all itsassociated components can be located on a rotatable element, such asrotor assembly. Further, some signal processing associated with detectorcan also be performed on the rotor assembly. For instance, a flow cell,its associated detectors and a processor for performing signalprocessing associated with the detector can be located on a rotatableelement, such as rotor assembly described with respect to FIGS. 2-9C ora bucket assembly described with respect to FIGS. 10A-D. In otherembodiments, a portion of the detector can be located on the rotatableelement and a portion can be located on a stationary off-rotor, such asthe flow cells and detectors described with respect to FIGS. 2-10D. Inyet other embodiments, a detector can be totally located off of therotatable element. For example, collected fractions can be transferredoff a rotatable element by some mechanism to a detector for furtheranalysis.

There are many types of detectors that can be used with embodimentsdescribed herein. Some of the detectors that be used with theembodiments described herein include: Refractive Index (RI),Ultra-Violet (UV), Visible, Fluorescent, Radiochemical, Electrochemical,Near-Infra Red (Near-IR), Mass Spectroscopy (MS), Nuclear MagneticResonance (NMR), Light Scattering (LS) and combinations thereof. Thesedetectors are described as follows.

There are two types of detectors in liquid chromatography. The firsttype can require direct contact of the detector's transducer with thecolumn eluent. These include electrochemical detectors of various typesand mass spectrometers. The second type does not require direct contactof any physical element of the detector with the column eluent. Thesecan include the detectors that measure the changes in one part oranother part of the electromagnetic spectrum as well as nuclear magneticresonance detectors.

Refractive Index (RI) detectors can be used to measure the ability ofanalyte molecules to bend or refract light. This property for eachmolecule or compound is called its refractive index. For some RIdetectors, such as differential RI detectors, light can proceed througha bi-modular flow-cell to a photodetector. One channel of the flow-cellcan direct the mobile phase passing through the column while the othercan direct only the mobile phase. Detection can occurs when the light isbent due to samples eluting from the column, which can be read as adisparity between the two channels. For other types of RI detectors thedegree to which the light is refracted by the analytes as they passthrough the flow cell can be detected directly without reference toanother flow cell.

Ultra-Violet (UV) and Visible (Vis) light detectors can be used measurethe ability of a sample to absorb light. This measurement can beaccomplished at one or several wavelengths. Typically, UV detectors canhave a sensitivity of about 10⁻⁸ or 10⁻⁹ gm/ml. Typically, UV detectorsrequire a light source and light gathering element. In general, lightsources can be broad spectrum lamps, emission line lamps, light emittingdiodes, or even a laser. As previously described, the light emitted fromthe light source can be directed through a flow cell or several opticalfocusing or filtering elements can be used to focus the light onto theoptically transparent window of the flow cell.

As examples, a fixed wavelength measurement can be used to detectabsorbance at one wavelength, such as 254 nm. A variable wavelengthmeasurement can be used to detect absorbance at different wavelengthsone wavelength at a time, but over a wide range of wavelengths. Forexample, for the embodiment where a flow cell rotates underneath astationary detector, a first wavelength of a light source can be used ona first revolution of a rotor assembly, a second wavelength on a secondrevolution and a third wavelength and then start over again with thefirst wavelength. This type of measurement could be repeated for many 2or more different wavelengths and is not limited to only 3 wavelengths.In another embodiment, a diode array can be used to measure a spectrumof wavelengths simultaneously. In yet another embodiment, severaldifferent light sources with or without accompanying optical focusingelements, mounted on a fixed position relative to the rotatable elementcan be focused onto the flow cell such that for each revolution of therotatable element, many different wavelengths can be used with a singlecommon flow cell on the rotatable element.

Scanning UV/Vis detectors can be used to measure and detect samples overthe entire UV to visible (UV-Vis) spectrum. They can be highly valuabletools in the identification and analysis of sample compounds. In thistype of detector, the response of the substance passing through the flowcell to the light in the flow cells is measured and recorded. To detectover an entire spectrum, the detector can proceed in one of two ways.The first can be to scan across the entire spectral region, which can beaccomplished by a scanning monochromator spectrometer. A standardscanning monochromator spectrometer can use a tungsten or deuterium lampthat emits a broad spectrum light source. The light can then be directedacross a grating or prism which reflects the light through an exit slitto the sample cell. The sample can then detected by a photomultipliertube. The wavelength of the light can be adjusted by rotating thegrating or prism, but only one region can be scanned at a time.Subsequently, data points can be obtained at different times.

The second method can involve monitoring the entire UV-Vis regionsimultaneously. One technique can be to use several photomultipliertubes positioned to detect in the spectral regions of interest. Anotheris to use the linear photodiode array (LPDA) spectrophotometer whichmeasures in the 190-1100 nm region simultaneously. No monochromaticlight is needed and data can be retrieved in milliseconds. Thisdetection methodology can be used for analyzing kinetic or chemicalintermediates, or separating and analyzing overlapping chromatographicpeaks using spectography.

Fluorescent detectors can be used measure the ability of a compound toabsorb light at a given wavelength (excitation) then re-emit light at aslightly higher wavelength. Each compound can have a characteristicfluorescence. The excitation source can pass through the flow-cell to aphotodetector while a monochromator measures the emission wavelengths.Typically, fluorescent detectors can have a sensitivity limit of about10⁻⁹ to 10⁻¹¹ gm/ml.

Laser-induced fluorescence (LIF) is a spectroscopic method that can beused for detecting species. As previously described, one or moredifferent LIF detectors can be used with the embodiments describedherein. The species to be examined can be excited with monochromaticlaser light. The wavelength can be often selected to be the one at whichthe species has its largest cross section. An excited species can, aftersome time, usually in the order of few nanoseconds to microseconds,de-excite and emit light at a wavelength larger than the excitationwavelength. The emitted light, fluorescence, can measured using adetector of some type.

In particular embodiments, disperse spectra and/or excitation spectracan be generated and measured. To perform a disperse spectrameasurement, a fixed lasing wavelength can be used and the generatedfluorescence spectrum can be analyzed. To perform an excitation spectrameasurement, a lasing wavelength can be varied and fluorescent light ata fixed emission wavelength or range of wavelengths can be measured forthe various lasing wavelengths.

One advantage fluorescent detectors is that it is possible to get two-and three-dimensional images since fluorescence takes place in alldirections (i.e. the fluorescence signal is isotropic). Further, thesignal-to-noise ratio of the fluorescence signal can be very high,providing a good sensitivity to the process. It can also be possible todistinguish between more species, since the lasing wavelength can betuned to a particular excitation of a given species which is not sharedby other species.

Radiochemical detection can involve the use of radio-labeled material,such as tritium (³H) or carbon-14 (¹⁴C). It can operate by detection offluorescence associated with beta-particle ionization. One applicationcan be metabolite research. Two detector types can be a) homogeneous andheterogeneous. In a homogeneous detector, a scintillation fluid can beadded to a column effluent to cause fluorescence. In a heterogeneousdetector, lithium silicate and fluorescence caused by beta-particleemission can interact with the detector cell. Typically, this type ofdetector can have sensitivity limit up to 10⁻⁹ to 10⁻¹⁰ gm/ml.

Electrochemical detectors can be used to measure compounds that undergooxidation or reduction reactions. Usually, this measurement can beaccomplished by measuring gain or loss of electrons from migratingsamples as they pass between electrodes at a given difference inelectrical potential. In particular embodiments, the electrodes can belocated near the end of one or more of the columns in the configurationspreviously described herein, such as on a rotor assembly or a bucketassembly. Typically, this type of detector can have a sensitivity limitof about 10⁻¹² to 10⁻¹³ gm/ml.

In a Mass Spectroscopy (MS) detector, a sample compound or molecule canbe ionized. Then, it can be passed through a mass analyzer, and the ioncurrent can be detected. There are various methods for ionization. In afirst method, often referred to as Electron Impact (EI), an electroncurrent or beam created under high electric potential can used to ionizethe sample migrating off the column. In a second method, often referredto as chemical ionization, ionized gas can be used to remove electronsfrom the compounds eluting from the column. In a third method, oftenreferred as Fast Atom Bombardment (FAB), xenon atoms can be propelled athigh speed in order to ionize the eluents from the column. This type ofdetector can have detector can have detection limit of 10⁻⁸ to 10⁻¹⁰gm/ml. In particular embodiments, this methodology can be performed on arotatable element during rotation of the element.

In Nuclear Magnetic Resonance (NMR) detectors, certain nuclei withodd-numbered masses, including H and ¹³C, can spin about an axis in arandom fashion. However, when placed between poles of a strong magnet,the spins can be aligned either parallel or anti-parallel to themagnetic field, with the parallel orientation favored since it isslightly lower in energy. The nuclei can then be irradiated withelectromagnetic radiation which is absorbed and places the parallelnuclei into a higher energy state; consequently, they are now in“resonance” with the radiation. Each H or C can produce differentspectra depending on their location and adjacent molecules, or elementsin the compound, because all nuclei in molecules are surrounded byelectron clouds which change the encompassing magnetic field and therebyalter the absorption frequency.

In light-scattering (LS) detectors, when a source emits a parallel beamof light which strikes particles in solution, some light can bereflected, absorbed, transmitted, or scattered. In Nephelometry, whichcan be defined as the measurement of light scattered by a particulatesolution, the detection of a portion of light scattered at a multitudeof angles can be detected. The sensitivity can depend on the absence ofbackground light or scatter since the detection occurs at a black ornull background.

In Turbidimetry, which can be defined as the measure of the reduction oflight transmitted due to particles in solution, the light scatter as adecrease in the light that is transmitted through the particulatesolution can be measured. Therefore, it can quantify the residual lighttransmitted. Sensitivity of this method can depend on the sensitivity ofthe machine employed, which can range from a simple spectrophotometer toa sophisticated discrete analyzer. Thus, the measurement of a decreasein transmitted light from a large signal of transmitted light can belimited to the photometric accuracy and limitations of the instrumentemployed.

Near-Infrared Detectors can operate by scanning compounds in a spectrumrange, such as from 700 to 1100 nm. The stretching and bendingvibrations of particular chemical bonds in each molecule can be detectedat certain wavelengths. With this type of detector, multiple analysescan be obtained from a single spectrum.

Chromatographic Separation

As described above, chromatography can be described as a process thatachieves physical separation of the individual components of a mixtureof chemical substances. In the chromatographic process, the mixture ofchemical substances can be dissolved in a carrier stream (gas orliquid). The carrier stream including the mixture can be forced througha bed of particles. The carrier stream moves at a velocity through thebed of particles. In chromatography, the carrier stream is oftenreferred to as the “mobile phase” and the bed of particles is referredto as the “stationary phase.”

In the embodiments previously described herein, chromatographicenclosures, such as cylindrical columns, are described that can containthe stationary phase for a chromatographic process. The chromatographicenclosure contains a chromatographic stationary phase. At least one flowpath is provided through the chromatographic enclosure such that fluidcan enter the chromatographic enclosure, pass through a chromatographicstationary phase and then exit the chromatographic enclosure.

In some embodiments, a number of separate flow paths are provided withinthe chromatographic enclosure where there is no fluid communicationbetween each of the separate flow paths. A separate chromatographicstationary phase is contained in each of the flow paths. The separateflow paths allow for “parallel” chromatographic processing where in eachof the flow paths, a common sample fluid or different sample fluids areprocessed in parallel.

A flow path is provided by a structure that includes a hollow innerportion. The fluid and the chromatographic stationary phase arecontained within the hollow inner structure. Generally, the fluid isdriven axially through the hollow portion of the structure. Forinstance, the structure can be a hollow pipe where the fluid is drivenaxially through the hollow pipe. A length, an inner cross section and aninner cross sectional area can be defined for hollow portion of thestructure associated with each flow path in the chromatographicenclosure. In some embodiments, the inner cross sectional area isconstant along the length of the flow path. In other embodiments, theinner cross sectional area varies along the length of the flow path. Insome embodiments, the inner cross section is circular. In general, theinner cross sectional geometry can be any shape.

In particular embodiments, an outer cross section of the structure issimilar to the inner cross section of the hollow portion of thestructure. For instance, the inner and outer cross section can becircles of different diameters, such that a cylindrical pipe is formed.In other embodiments, the inner and outer cross sections are notsimilar. For instance, a circular hollow tube can be drilled into arectangular block of material. In some embodiments, the chromatographicenclosure is integrally formed with a rotor or housing separate from therotor.

Each chromatographic enclosure includes an entrance and an exit. Theentrance allows fluid to enter one or more separate flow paths. The exitallows fluid to exit from one or more separate flow paths. A fluid, suchas the mobile phase of a chromatographic process, can enter via theentrance and exit via the exit to establish a flow of the fluid throughthe chromatographic enclosure including a flow through thechromatographic stationary phase contained within the enclosure.

The chromatographic enclosures can be carried by a rotor that provides acentrifugal force to the chromatographic enclosure. For instance, asdescribed above, the chromatographic enclosures can be carried on arotor assembly that is arranged to rotate at an angular velocity. Thecentrifugal forces provide a driving a driving force for moving fluidthrough the chromatographic enclosure. The fluid can include a sample.As the fluid including the sample moves through the stationary phasecontained in the chromatographic enclosure a chromatographic separationof the sample can occur.

As an example, FIG. 11 is a front view of a chromatographic enclosureconfigured for a chromatographic process before and aftercentrifugation. A column, such as a cylindrical plastic syringe 600, canbe secured within a container, such as the centrifuge tube 604. Theplastic syringe 600 can include a top opening 615 and a bottom opening616. In this embodiment, the area of the of column changes over itslength as the top opening 615 is larger than the bottom opening 616. Aspreviously described, in other embodiments, the column can be a constantarea over its length.

To set-up a chromatographic process, prior to centrifugation, a numberof components can be added to the plastic syringe 600. A porous plug,such as glass wool 616, can be added near the bottom of the syringe 600.The porous plug can prevent a solid stationary phase, such as 610, fromexiting the column but allow fluids to exit the column.

In one embodiment, the stationary phase can be a chromatographicadsorbent 610. The type of stationary phase that is employed can varydepending on a type of chromatographic process that is set-up. A samplecan interact differently with a stationary phase material depending on atype of solvent and a type of stationary phase material that is used.Different types of chromatographic processes and a few characteristicsof the stationary phase materials and solvents that are used in theseprocesses are described below, following the description of FIG. 11.

A sample 608, which can be dissolved in a liquid, can be placed abovethe stationary phase. The size of the stationary phase particles can beselected such that the sample does not penetrate the stationary phaseunder the force of gravity alone. A porous separator, such as sand 606,can be placed above the sample 608. The separator can separate thesample from a solvent source, such as solvent reservoir 602. The solventsource can act as a mobile phase in the chromatographic process. It canbe placed above the sample and the separator 606.

Next, the chromatographic process configuration can be coupled to adevice that can impart an angular velocity to the configuration, such asone of the rotor assemblies previous described. Under centrifugation,the solvent in the solvent reservoir 602 and the sample 608 can bedriven into the stationary phase, such as 610 and down the length of thecolumn. The sample 608 and the solvent from the solvent reservoir 602can move through the stationary phase during centrifugation. After aperiod of time, the sample 608 may have moved some distance throughstationary phase 610. Thus, the stationary phase is shown above andbelow the sample 608. Also, a portion of the mobile phase may havepassed entirely through stationary phase 610 and out the exit 616 of thecolumn to collect, as eluent 618, in a bottom of the centrifuge tube604.

This embodiment is provided for the purposes of illustration only and isnot meant to be limiting. In the embodiment of FIG. 11, the sample canbegin to move through the stationary phase prior to a steady flow beingestablished in the column. Thus, chromatographic separation can beginprior to a steady flow being established. In previously describedembodiments, a steady flow can be established in the column prior tosample introduction.

In the embodiment of FIG. 11, a solvent composition, which can be amixture of different solvents, can be used in the solvent reservoir 602.During centrifugation, the solvent reservoir 602 is not replenished andthe composition of the solvent is not changed. In previously describedembodiments, during centrifugation, the solvent reservoir 602 can bereplenished and the composition of the solvent can be changed, i.e.,gradient elution can be employed.

Different types of chromatographic processes can be performed dependingon a composition of the mobile phase and a composition of the stationaryphase. The embodiments described herein are applicable to any type ofchromatographic process where a mobile phase is moved through astationary phase in a chromatographic enclosure. A few examples ofchromatographic processes in which the method and apparatuses describedherein can be utilized include but are not limited to partitionchromatography, adsorption (liquid-solid) chromatography, ion exchangechromatography, affinity chromatography and size exclusionchromatography, such as gel permeation or gel filtration. Partitionchromatography generally includes both bonded phase and adsorbed phasereversed phase partition chromatography as well as normal partitionchromatography.

In adsorption chromatography, a mobile liquid or gaseous phase can beused that is adsorbed onto the surface of a stationary solid phase. Thedifferential equilibration between the mobile and stationary phaseaccounts for the separation of different solutes. In partitionchromatography, a thin film can be formed on the surface of a solidsupport by a liquid stationary phase. The thin film can be covalentlybound onto the surface of the support particle (bonded phase) oradsorbed onto the surface of the support particle (non-bonded phase).Analytes can differentially equilibrate between the mobile phase and thestationary liquid. In Ion-Exchange (IEX) Chromatography, solute ions ofthe opposite charge in the mobile liquid phase are attracted to theresin (or particulate stationary phase) by electrostatic forces and thegreater the charge of the analyte, the more the analyte interacts withthe surface of the stationary phase particles and the longer it takes totraverse the chromatographic system. IEX chromatography can be alsouseful for determining the tertiary structure and quaternary structureof purified proteins, especially since it can be carried out undernative solution conditions using only aqueous solutions.

In size exclusion chromatography, an attractive interaction between thestationary phase and solute is not used. Instead, the liquid or gaseousphase passes through a porous gel which separates the moleculesaccording to its size. The pores are normally small and exclude thelarger solute molecules, but allow smaller molecules to enter the gel,causing them to flow through a larger volume. This causes the largermolecules to pass through the column at a faster rate than the smallerones.

In affinity chromatography, the specific interaction between one kind ofsolute molecule and a second molecule that is immobilized on astationary phase can be utilized. For example, the immobilized moleculemay be an antibody to some specific protein. When a solute containing amixture of proteins are passed by this molecule, only the specificprotein is reacted to this antibody, binding it to the stationary phase.This protein can later be extracted by changing the ionic strength orpH.

In more detail, Reverse Phase Partition Chromatography (RPC) can includeany chromatographic method that uses a non-polar stationary phase. Itdiffers from “normal” partition chromatography (NPC) which can be doneon non-modified silica or alumina with a hydrophilic surface chemistryand a stronger affinity for polar compounds. In RPC, the introduction ofalkyl chains bonded covalently to the support surface can reverse theelution order as compared to NPC. In RPC, polar compounds can be elutedfirst while non-polar compounds are retained—hence it is called“reversed phase.”

For RPC, any inert non-polar substance that achieves sufficient surfacecoverage of the particles of the packing can be used. One column can bean octadecyl carbon chain (C18) bonded silica (USP classification L1),297 columns are commercially available. Another column can be by C8bonded silica (L7—166 columns are commercially available). Other columnscan be cyano bonded silica (L10—73 columns are commercially available)and phenyl bonded silica (L11—72 columns are commercially available).C18, C8 and phenyl are dedicated reversed phase packings while cyanocolumns can be used in a reversed phase mode depending on analyte andmobile phase conditions. It should be noted at this point that not allC18 columns have identical retention properties. Surfacefunctionalization of silica can be performed in a monomeric or apolymeric reaction with different short-chain organosilanes used in asecond step to cover remaining silanol groups (end-capping). While theoverall retention mechanism remains the same subtle differences in thesurface chemistries of different stationary phases will lead to changesin selectivity.

In RPC, mixtures of water (or buffered aqueous solutions) and organicsolvents can be used to elute analytes from a reversed phase column. Thesolvents can be miscible with water and the most common organic solventsused are acetonitrile, methanol or tetrahydrofuran (THF). Other solventscan be used such as ethanol and 2-propanol (isopropyl alcohol). Elutioncan be performed isocratically (the water-solvent composition does notchange during the separation process) or by using a gradient (thewater-solvent composition does change during the separation process).The pH of the mobile phase can have an important role on the retentionof an analyte and can change the selectivity of certain analytes.Charged analytes can be separated on a reversed phase column by the useof ion-pairing (also called ion-interaction). This technique is known asreversed phase ion-pairing chromatography.

Sample Results and Comparison with HPLC

An effectiveness of chromatographic separation process is often referredto as a “chromatographic efficiency.” Chromatography efficiency theorydescribes parameters that allow relative efficiencies of variouschromatographic processes to be compared. It is believed that utilizingthe methods and apparatus described herein, chromatographic processescan be set-up that are more “efficient” than similar chromatographicprocesses that can be performed using other types of apparatus andmethods, such as chromatographic processes using HPLC. Thus, in thefollowing paragraphs, methods and apparatus for centrifugal liquidchromatography including pressure requirements and particle sizelimitations are compared HPLC, which is a commonly practiced form ofliquid chromatography. Further, a brief description of chromatographicseparation theory is described including 1) measure performancemeasurements of chromatographic separation efficiencies using apparatusand methods described herein and 2) a comparison of the estimatedperformance measurements with performance measurements obtained withother types of chromatographic apparatus, such as HPLC.

In HPLC, a common quantity that is employed is the back pressure. A backpressure calculation can be used to determine how much pressure isneeded to force a fluid through a stationary phase in a column where thestationary particles are a particular size. The required pressure inHPLC can be calculated as,

ΔP=(ηFL)/(K ⁰ πr ² d _(P) ²)

where ΔP is the pressure at the column head which decreases across thecolumn length, η is the viscosity, F is the flow rate, L is the lengthof the column, K⁰ is the specific permeability, r is the column radiusand d_(P) is the particle diameter. It can be seen that increasing theflow rate, a fluid viscosity and length of the column or decreasing theparticle diameter all lead to greater pressure requirements. For smallparticle sizes, such as below 2 micrometers, in HPLC, the pressurerequirements become prohibitive. For example, in HPLC, pressures above10,000 PSI can be required to utilize 2 micrometer particles.

In the embodiments described herein using centrifugal liquidchromatography, the backpressure requirements and an importance ofpressure appear to be different than HPLC. For instance, for a givenparticle size as well as the other parameters in the formula describedabove, the pressure at which the chromatographic systems describedherein operate appears to be much less than what would be required inHPLC system. Also, systems using centrifugal liquid chromatography canbehave differently in regards to pressure than HPLC systems. Forinstance, in some embodiments described herein, the pressure canincrease from the head of the column along the column length rather thandecrease along the column like in HPLC.

Without being bound by a particular theory, the column can be viewed asa number of layers where a fluid moves from layer to layer as itproceeds down the column. In separation efficiency theory, as isdescribed below, each of these layers can be referred to as a “plate.”When pressure is used as a driving force, such as in HPLC, it isbelieved a small amount of pressure is needed to move the fluid acrosseach layer. Thus, the required pressure depends on the number of layersor plates in the column where the total required pressure can beconsidered as the summation of the pressures required to move the fluidacross each of the layers. Separation efficiency is usually increased asthe number of layers increase. Thus, in HPLC, increasing the number oflayer and hence the separation efficiency can require more pressure.

In centrifugal liquid chromatography, the fluid can be moved across eachlayer via centrifugal forces and not a drop in pressure. The centrifugalforces can act on each layer independently of one another withoutnecessarily requiring a pressure drop across the layer to move the fluidacross the layer as in HPLC. Thus, in centrifugal liquid chromatography,the pressure requirements appear to much less than that of HPLC.Applicant believes this feature may not have been fully appreciated inthe prior art.

Further, it is believed in the embodiments described herein much smallerparticles can be used than are possible with HPLC. For instance, a useof particle sizes of about 15 angstroms may be possible. If smallerparticles can be manufactured, than it is believed that smaller sizeparticles can be used.

In chromatography, a plate model can be used to estimate separationefficiencies. The plate model supposes that the chromatographic columncontains a large number of separate layers, called “theoretical plates.”Separate equilibrations of the sample between the stationary and mobilephase can occur in these “plates”. The analyte moves down the column bytransfer of equilibrated mobile phase from one plate to the next.

The term “plate” is used as an analogy for the processes at work in thecolumn as “plates” do not actually exist in the column. The columnefficiency, i.e., its ability to perform chromatographic separation canbe quantified by determining a number of theoretical plates in thecolumn, N, or by stating the plate height, which is often referred to asHeight Equivalent to a Theoretical Plate (HETP). For column of length,L, HETP can be defined as,

HETP=L/N.

When comparing chromatographic processes occurring in two differentcolumns, a larger value of N or a smaller value of HETP for a firstcolumn as compared to a second column can indicate that the first columnhas a greater chromatographic efficiency than the second column.

A definition of separation efficiency has been developed for liquidchromatography taking place in a column. The International Union of Pureand Applied Chemistry defines separation efficiency as:

N=16(V _(R) /w _(b))²=16(t _(r) /w)²

where N is the number of theoretical plates, V_(R) is the volume ofmobile phase entering the column between sample injection and theemergence of the peak maximum of the sample component of interest, w_(b)is the total volume from the time the analyte begins emerging from thecolumn to the time it has completely left the column or t is theretention time (seconds) and w is the width of the peak (seconds).

Experimental Measurements were made using a chromatographic system forone embodiment described herein. For a separation of three species, acolumn length of 2.3 cm, packed with a particle size of diameter of 5micrometers, (d_(p)) and a void volume of 0.110 ml is utilized. Species1 is FD&C Red No. 3 (Erythrosine). Species 2 is FD&C Yellow No. 5(Tartrazine). Species 3 is FD&C Green No. 3 (Fast Green FCF, E143). Themobile phase is 70% Water, 30% Isopropyl Alcohol (IPA), and 0.010 molarTetrabutylammonium Phosphate (TBAP). In this experiment, a theoreticalplate height is generated. These experiments resulted in the followingvalues as shown in Table 1.

TABLE 1 Experimentally Measured Values α R (1, 2) (1, 2) Spe- V_(R)w_(b) (1, 3) (1, 3) N N/L cies (ml) (ml) k′ (2, 3) (2, 3) (Column)(meter) 1 5.3 .009 47.2 1.15 94.1 5,548,642 2,412,453,033 2 6.1 .00854.5 1.42 244.4 9,302,500 4,044,565,217 3 7.5 .009 67.2 1.23 164.711,111,111 4,830,917,874

V_(R) and w_(b), which is a value for each species, are described abovewith regard to the theoretical plate formula. k′ is a capacity factorfor each species, α is the ratio of retention volumes between twospecies and R is a resolution between two species, N is the number oftheoretical plates and N/L is the number of theoretical platesnormalized by the length of the column (0.023 m). Resolution between twospecies, such as between species 1 and 2, described above can becalculated as R(A,B)=2 [V_(R-B-)V_(R-A)]/[w_(b-B)+w_(b-A)]. For example,R(1,2)=[V_(R-2-)V_(R-1)]/[w_(b-2)+w_(b-1)]. α(A,B)=k′_(A)/k′_(B) is aratio of capacity factors between two species. For instance,α(1,2)=k′₁/k′₂.

A chromatogram with the 3 species described in Table 1 is shown in FIG.12. The quantities derived in Table, such as theoretical plates, can bederived from data obtained from the chromatogram, such as a Volume ofeach peak. Although appearing as lines, on the smaller scale, the peakshave a width and can be used to calculate a volume.

The theoretical plates for each species and resolution between speciesare quite high. For instance, a resolution value between two species of1.5 or greater is considered completely separated. The theoretical platevalues and resolution values appear to be much higher than theoreticalplate values and resolution values obtained using HPLC.

To quantify the differences between HPLC and the methods and apparatusdescribed herein, a comparable separation was performed in HPLC and thenusing centrifugal liquid chromatography. Each separation used 5micrometer diameter particles in the stationary phase. A length of thecolumn used in the HPLC was 150 mm. A length of the column used with thecentrifugal liquid chromatography was 36 mm. A similar mobile phasefluid and sample were used in both methods. Measured results for theexperiments are shown in Table 2.

TABLE 2 Experimentally Measured Values N N/L Methodology (Column)(meter) HETP HPLC 9,604 64,027 16 micrometers Centrifugal Liquid17,650,317 490,286,583 about 2 nanometers Chromatography

The large differences between HPLC and the centrifugal liquidchromatography measurement were unexpected. The centrifugal liquidchromatography measurements indicate separation efficiencies that areorders of magnitude better than HPLC. There are number of possibleexplanations for the increased separation efficiencies of centrifugalliquid chromatography. Without being bound by a particular theory as towhy the exemplary results are obtained, a few observations can be madeas follows.

Diffusion resulting from concentration gradients appears to be muchlower when centrifugation is used to drive a flow through achromatographic enclosure as compared to using pressure as in HPLC.Reduced diffusion can increase separation efficiency hence contribute tothe large number of theoretical plates obtained with the embodimentsdescribed herein as opposed to HPLC. Sedimentation coefficientsassociated with centrifugation are concentration dependent. This effectmay contribute to a decrease in diffusion obtained with centrifugation.

Applications

Chromatography is a fundamental tool for practicing chemists as well asothers who are applying chemistry in their own discipline.Chromatography is widely used in and essential to the petroleumindustry, food industry, pharmaceutical industry, medicine (e.g.,diagnostics), and many others. In more detail, the embodiments describedherein can be used for chromatographic processes applied to:

Medical and biomedical research

Quality control of pharmaceuticals

Routine clinical determination

Drug screening

Space-related research and development

Geochemical research and development

Pharmaceutical research and development

Forensic sciences

Food and cosmetic chemical measurement

Process control in the petroleum industry

Environmental monitoring and pollution control

Investigation of the chemistry and metabolism of biological systems

A few specific types of analyses that can be performed using the devicesdescribed herein include but are not limited to the analysis of humanplasma proteins, nucleotides and their derivatives, amino acids andtheir derivatives, urinary metabolites, therapeutic drug monitoring,monitoring for drugs of abuse, and the analysis of the proteins of wheatand other seeds.

Two main goals of a chromatographic process, distinguished by theirscale and purpose, can be analytical and preparative. Preparativechromatography can be used for large scale recovery and purification ofa few samples. Analytical chromatography can be used to the small scaleanalysis of many samples to determine their composition and purity. Afew examples of the samples size, column length and column innerdiameter (I.D.) that can be involved in different preparative andanalytical applications are described as follows,

Column Column Name Purpose Scale Length I.D. Micro- Data and somelimited nanograms to 10-400 cm .1-2 mm Analytical compound collectionmicrograms Analytical Data and some limited micrograms to  10-25 cm 2-5mm compound milligrams collection(compound identification andconcentration) Semi- Data and a small amount a few milligrams 10-100 cm5-25 mm preparative of purified compound to a few hundred milligramsPreparative Larger amounts of a few hundred 50-200 cm 25-100 mm purifiedcompound milligrams to a few grams Process (Industrial) a few grams to a100-250 cm  100-2000 mm Manufacturing quantities several kilogramsThe embodiments described herein can be used for both preparative andanalytical applications involving sample sizes that vary and scaleaccordingly, such as the applications described above. In situationsinvolving larger samples, such as industrial processes, the apparatusand methods can be scaled up according. Column sizes, such as columnlength and a column I.D., which can vary depending on the application,as shown above, can range from 10-400 cm and 0.1-2000 mm, respectively,in various embodiments. The column length and I.D. can be configured tomany different sizes to satisfy the requirements of a particleapplication. For instance, capillary tubes with a I.D. of 0.01 mm orless can be used. Thus, the ranges described above are not meant tolimiting and are provided for the purposes of illustration only.Rotatable elements, such as rotor assemblies described above, can bescaled up or down, to accommodate columns of a particular size that areutilized in a particular application.

Methods

FIG. 13 is a flow chart of a method 700 for performing a chromatographicseparation process. In 702, a number of components, such as 1) achromatographic enclosure including a stationary phase, 2) a mobilephase fluid source and 3) a sample source can be provided. In 704, thechromatographic enclosure can be coupled to a rotatable element, such asa rotor assembly.

In 706, the rotatable element can be rotated from rest such that anangular velocity is imparted to the chromatographic enclosure. Duringrotation, a mobile phase fluid from the mobile phase fluid source can bemoved through the stationary phase via centrifugal force. The mobilephase fluid source can be an integral component of the chromatographicenclosure or can be located separately from the chromatographicenclosure.

In 708, during rotation of the chromatographic enclosure, it can bedetermined whether a flow of the mobile phase fluid through thestationary phase is established. In particular, it can be determinedthat a steady flow is established through the chromatographic enclosure.In one embodiment, measurements made using a flow cell located near theend of the chromatographic enclosure can be used to determine whetherthe flow is established.

In general, a detector can be used to determine whether a steadycondition has been reached in a column. Examples of detectors that canbe utilized are described above, such as the detectors described in theinstrumentation section. A signal from the detector can be examined oversome time period, such as the signal generated from a mobile phase fluidleaving the column. The chromatogram shown in FIG. 12 is an example of asignal that can be obtained from a detector. Some noise can beassociated with the signal. Steadiness can be defined as the averagevalue of the signal varying less than some amount over a time period ofinterest. Multiple detectors can be associated with a column and signalsfrom multiple detectors can be used to determine whether a steadycondition has been reached.

Another factor that can be examined is the angular velocity of the rotorassembly. In some embodiments, the rotor assembly can be spun up fromrest to a target angular velocity for a particular run or the rotorassembly can be at a first angular velocity for a first run and then itsvelocity can be increased or decreased to a new target value for asubsequent run. A detector can be used to determine whether the averageangular velocity of the rotor assembly is within some range over sometime period. For instance, a motor associated with the rotor assemblycan report its speed. Until the average angular velocity is determinedto be in range over a time period of interest, in some embodiments,sample injection may not proceed. In some embodiments, prior toinitiating sample injection, a combination of signals from one or moredetectors associated with a flow through a column and a signalassociated with an angular velocity may both have to be within a rangeover a time period.

As an example, a series of runs on a chromatographic system can involvefirst introducing a mobile phase fluid into a column. The composition ofthe mobile phase fluid can produce a first signal that is output fromthe detector. When this signal is varying within a certain range overtime, such as the signal value level is relatively flat, a sample can beinjected and a gradient elution can be performed. In some instances, thegradient elution can be required to allow certain analytes to leave thecolumn. The gradient elution can produce a signal change output from thedetector. The signal change resulting from elution can have arecognizable profile, such as a line with a particular slope. Also,analytes leaving the column can produce a signal change. The signalchange from the analytes can have a recognizable profile, such as peaksand valleys. The signal change produced by the analytes can have aprofile that differs from the signal change produced by gradientelution, which allows in some instances, their individual contributionsto the signal change to be recognized.

After the gradient elution is completed, the mobile phase fluid can bereturned to its initial composition prior to the gradient elution. Inresponse, the signal from the detector can return to its initial valueof after time period, i.e., the value before gradient elution began. Insome embodiments, 10-20 column volumes of fluid may be allowed toproceed through the column. When the signal returns to its initial valueand its value is within some range over some time period, then, thecolumn can be considered ready for another sample injection.

In 710, during rotation of the chromatographic enclosure, a sample fluidfrom the sample source can be introduced such that the sample fluid canmove through the stationary phase. The movement of the sample fluidthrough the stationary phase can result in a chromatographic separationof one or more sample components from the sample fluid. In 712, duringrotation of the chromatographic enclosure, a separated component of thesample fluid after it has passed pass through the stationary phase canbe detected. For example, the separated component can pass through aflow cell near the end of the chromatographic enclosure wheremeasurement performed using the flow cell can be used to detect apresence of the separated component. The measurement can be displayed asa chromatogram on an output device associated with the chromatographicsystem.

The advantages of the invention are numerous. Different aspects,embodiments or implementations may yield one or more of the followingadvantages. One advantage is that using centrifugation to drive fluidthrough a chromatographic column can allow smaller particle stationaryphase particles to be used than other types of chromatographicmethodologies, such as HPLC. The use of smaller particles can providefor greater chromatographic separation efficiencies than are possiblewith other types of chromatographic methodologies, such as HPLC. Anotheradvantage is that the rotor assemblies described herein can allow for alarge number of chromatographic processes to be performedsimultaneously. The “parallel processing” of a large number ofchromatographic processes can provide for shorter throughput times thatenable analyses that are too time consuming and cost prohibitive to becarried out with other chromatographic processes, such as HPLC, to beperformed. The many features and advantages of the present invention areapparent from the written description and, thus, it is intended by theappended claims to cover all such features and advantages of theinvention. Further, since numerous modifications and changes willreadily occur to those skilled in the art, the invention should not belimited to the exact construction and operation as illustrated anddescribed. Hence, all suitable modifications and equivalents may beresorted to as falling within the scope of the invention.

1. A centrifugal chromatographic system comprising: a chromatographicenclosure; a rotor that carries the chromatographic enclosure whereinthe rotor is configured to rotate the chromatographic enclosure; asample introduction mechanism in fluid communication with thechromatographic enclosure, said sample introduction mechanism beingarranged to introduce a sample fluid to the chromatographic enclosurewhile the rotor is rotating, wherein the sample introduction mechanismis configured to receive a sample introduction signal and to trigger theintroduction of the sample fluid in response to receiving the sampleintroduction signal.
 2. A centrifugal chromatographic system as recitedin claim 1 further comprising a controller arranged to automaticallytrigger the introduction of the sample fluid into the chromatographicenclosure.
 3. A centrifugal chromatographic system as recited in claim 1further comprising a controller, coupled to the sample introductionmechanism, wherein the controller is designed or configured to 1) toreceive a signal from a detector mechanism, 2) to determine that achange in the signal is within a range over a time period; 3) when thesignal is within the range over the time period, instruct the sampleintroduction mechanism to release the sample fluid.
 4. A centrifugalchromatographic system as recited in claim 3, wherein the signal fromthe detector mechanism is associated with a characteristic of the fluidthat has passed through the chromatographic enclosure.
 5. A centrifugalchromatographic system as recited in claim 3, wherein detector mechanismconfigured to generate a signal associated with the angular velocity ofthe rotor.
 6. A centrifugal chromatographic system as recited in claim1, further comprising a controller, coupled to the sample introductionmechanism, wherein the controller is designed or configured to 1) todetermine whether steady conditions are established in thechromatographic enclosure; 2) based at least in part upon thedetermination of whether steady conditions are established, instruct thesample introduction mechanism to release the sample fluid.
 7. Acentrifugal chromatographic system as recited in claim 6, furthercomprising a detector mechanism, communicatively coupled to thecontroller, configured to generate a signal associated with an operatingcharacteristic of the chromatographic system that changes over time;wherein the determination by the controller of whether steady conditionsare reached is based at least in part upon signal received from thedetector mechanism.
 8. A centrifugal chromatographic system as recitedin claim 1, further comprising a gradient former for providing a mobilephase fluid comprising two or more different fluids wherein the gradientformer is arranged to vary over time a percentage of each of the two ormore different fluids delivered to the chromatographic enclosure.
 9. Acentrifugal chromatographic system as recited in claim 1, wherein therotor carries a plurality of chromatographic column enclosures.
 10. Acentrifugal chromatographic system as recited in claim 1, wherein thechromatographic enclosure is arranged to contain an associatedchromatographic stationary phase and to facilitate transmission of afluid through the chromatographic stationary phase contained within thechromatographic enclosure wherein the fluid is driven through thechromatographic stationary phase via centrifugal force generated fromthe rotation of the rotor.
 11. A centrifugal chromatographic system asrecited in claim 1, further comprising a mobile phase fluid compatiblewith a chromatographic process wherein the chromatographic processincludes one or more of reversed-phase partitioning, adsorption, anionexchange, cation exchange, size exclusion, gel filtration, affinityinteractions or combinations thereof.
 12. A centrifugal chromatographicsystem comprising: a chromatographic enclosure; a rotor that carries thechromatographic enclosure wherein the rotor is configured to rotate thechromatographic enclosure; a sample introduction mechanism in fluidcommunication with the chromatographic enclosure the sample introductionmechanism being arranged to introduce a sample fluid to thechromatographic enclosure while the rotor is rotating; and a controller,coupled to the sample introduction mechanism and arranged toautomatically trigger the introduction of the sample fluid.
 13. Acentrifugal chromatographic system as recited in claim 12 wherein thecontroller is arranged to trigger the introduction of the sample fluidbased at least in part on a sensed condition associated with thecentrifugal chromatographic system.
 14. A method of operating acentrifugal chromatographic system comprising: rotating a rotor thatcarries a chromatographic column; establishing a flow of a mobile phasefluid through the chromatographic column in which centrifugal forcedrives the mobile phase through the chromatographic column; andintroducing a sample fluid into the chromatographic column by insertingthe sample fluid into the mobile phase fluid flow, wherein theintroduction of the sample occurs while the rotor rotates.
 15. A methodas recited in claim 14, further comprising triggering the release of thesample fluid.
 16. A method as recited in claim 14, further comprising:determining whether a steady mobile phase fluid flow condition has beenreached within the chromatographic column; and wherein the sample fluidis released after it is determined that a steady mobile phase fluid flowcondition has been reached within the chromatographic column.
 17. Themethod of claim 16, further comprising determining whether the steadymobile phase fluid flow condition has been reached based upon a changein a physical characteristic over time of a fluid exiting thechromatographic column.
 18. The method of claim 17, wherein the physicalcharacteristic is measured using a flow cell proximate to the exit ofthe chromatographic column.
 19. A method of operating a centrifugalchromatographic system comprising: rotating a rotor that carries aplurality of chromatographic columns that each contain a chromatographicstationary phase; and while continuously rotating the rotor, passingthrough at least one of the chromatographic columns at least twodifferent samples wherein each of the samples is introduced at differenttimes.
 20. A method of operating a centrifugal chromatographic system asrecited in claim 19, wherein 10-20 column volumes of fluid are passedthrough the one chromatographic column between an introduction of afirst sample and an introduction of a second sample.
 21. A method ofoperating a centrifugal chromatographic system comprising: rotating arotor that carries a chromatographic column in which centrifugal forcedrives a mobile phase fluid through the chromatographic column; andtriggering a release of a sample fluid in which centrifugal force drivesthe sample fluid through the chromatographic column to facilitatecentrifugal chromatographic separation of the sample fluid, wherein therelease of the sample fluid occurs during rotation of the rotor.
 22. Amethod of operating a centrifugal chromatographic system comprising:rotating a rotor that carries a chromatographic column that contains achromatographic stationary phase; delivering a mobile phase fluid to thechromatographic column while the rotor rotates so that centrifugal forcedrives the mobile phase fluid through the chromatographic stationaryphase; and determining when to introduce a sample fluid into thechromatographic column to facilitate centrifugal chromatographicseparation of the sample fluid.
 23. A method of operating a centrifugalchromatographic system comprising: providing a rotor configured torotate about an axis and a chromatographic column arranged to contain achromatographic stationary phase carried on the rotor; rotating therotor and the chromatographic column to drive, via centrifugal force, amobile phase fluid through the chromatographic stationary phase;determining whether a flow of the mobile phase fluid is steady; and inresponse to the determination of whether the flow is steady, introducinga sample fluid wherein the sample fluid is driven throughchromatographic column via the centrifugal force.